Vaccination and methods against diseases resulting from pathogenic responses by specific T cell populations

ABSTRACT

The present invention provides vaccines and a means of vaccinating a vertebrate so as to prevent or control specific T cell mediated pathologies, including autoimmune diseases and the unregulated replication of T cells. The vaccine is composed of a T cell receptor (TCR) or a fragment thereof corresponding to a TCR present on the surface of T cells mediating the pathology. The vaccine fragment can be a peptide corresponding to sequences of TCRs characteristic of the T cells mediating said pathology. Such a peptide can bind to conventional antigens complexed to MHC antigen presenting cells or to superantigens. Means of determining appropriate amino acid sequences for such vaccines are also provided. The vaccine is administered to the vertebrate in a manner that induces an immune response directed against the TCR of T cells mediating the pathology. This immune response down regulates or deletes the pathogenic T cells, thus ablating the disease pathogenesis. The invention additionally provides specific β-chain variable regions of T cell receptors, designated Vβ3, Vβ4, Vβ12, Vβ14 and Vβ17, which are associated with the pathogenesis of autoimmune diseases, such as rheumatoid arthritis (RA) and multiple sclerosis (MS). Also provided are means to detect, prevent and treat RA and MS. Methods of administering DNA or RNA encoding the polypeptides useful as vaccines of the present invention into the tissue cells of an individual is also provided.

This application is a continuation of U.S. Ser. No. 07/813,867 filedDec. 24, 1991, now abandoned, which is a continuation-in-part of U.S.Ser. No. 07/644,611 filed Jan. 22, 1991, now abandoned, which is acontinuation-in-part of U.S. Ser. No. 07/530,229 filed May 30, 1990, nowabandoned, which is a continuation-in-part of U.S. Ser. Nos. 07/382,085and 07/382,086, both filed Jul. 18, 1989, both now abandoned, arecontinuations-in-part of U.S. Ser. No. 07/382,314, Mar. 21, 1989, nowabandoned. The contentsof all such related applications are incorporatedherein by reference in their entirety.

This invention is a continuation-in-part of copending U.S. Ser. No.07/644,611, filed Jan. 22, 1991, which is a continuation-in-part ofcopending U.S. Ser. No. 07/530,229, filed May 30, 1990, which is acontinuation-in-part of copending U.S. Ser. Nos. 07/382,085 and07/382,086, both filed on Jul. 18, 1989, which are continuations-in-partof copending U.S. Ser. No. 07/326,314, filed Mar. 21, 1989. The contentsof all such related applications are incorporated herein by reference intheir entirety.

BACKGROUND OF THE INVENTION

This invention relates to the immune system and, more specifically, tomethods of modifying pathological immune responses.

Higher organisms are characterized by an immune system which protectsthem against invasion by potentially deleterious substances ormicroorganisms. When a substance, termed an antigen, enters the body,and is recognized as foreign, the immune system mounts both anantibody-mediated response and a cell-mediated response. Cells of theimmune system termed B lymphocytes, or B cells, produce antibodies thatspecifically recognize and bind to the foreign substance. Otherlymphocytes termed T lymphocytes, or T cells, both effect and regulatethe cell-mediated response resulting eventually in the elimination ofthe antigen.

A variety of T cells are involved in the cell-mediated response. Someinduce particular B cell clones to proliferate and produce antibodiesspecific for the antigen. Others recognize and destroy cells presentingforeign antigens on their surfaces. Certain T cells regulate theresponse by either stimulating or suppressing other cells.

While the normal immune system is closely regulated, aberrations inimmune response are not uncommon. In some instances, the immune systemfunctions inappropriately and reacts to a component of the host as if itwere, in fact, foreign. Such a response results in an autoimmunedisease, in which the host's immune system attacks the host's owntissue. T cells, as the primary regulators of the immune system,directly or indirectly effect such autoimmune pathologies.

Numerous diseases are believed to result from autoimmune mechanisms.Prominent among these are rheumatoid arthritis, systemic lupuserythematosus, multiple sclerosis, Type I diabetes, myasthenia gravisand pemphigus vulgaris. Autoimmune diseases affect millions ofindividuals world-wide and the cost of these diseases, in terms ofactual treatment expenditures and lost productivity, is measured inbillions of dollars annually. At present, there are no known effectivetreatments for such autoimmune pathologies. Usually, only the symptomscan be treated, while the disease continues to progress, often resultingin severe debilitation or death.

In other instances, lymphocytes replicate inappropriately and withoutcontrol. Such replication results in a cancerous condition known as alymphoma. Where the unregulated lymphocytes are of the T cell type, thetumors are termed T cell lymphomas. As with other malignancies, T celllymphomas are difficult to treat effectively.

Thus, a long-felt need exists for an effective means of curing orameliorating T cell mediated pathologies. Such a treatment shouldideally control the inappropriate T cell response, rather than merelyreducing the symptoms. The present invention satisfies this need andprovides related advantages as well.

SUMMARY OF THE INVENTION

The present invention provides vaccines and a means of vaccinating avertebrate so as to prevent or control specific T cell mediatedpathologies. The vaccine is composed of a substantially pure T cellreceptor (TCR) or an immunogenic fragment thereof corresponding to a TCRpresent on the surface of T cells mediating the pathology. The vaccinefragment can be a peptide corresponding to sequences of TCRscharacteristic of the T cells mediating said pathology.

The invention additionally provides specific β-chain variable regionsand their immunogenic segments, and in particular three T cellreceptors, designated Vβ3, Vβ14 and Vβ17, which are associated with thepathogenesis of autoimmune diseases, for example rheumatoid arthritis(RA) and multiple sclerosis (MS). Additional VDJ junctional (CDR3)regions associated with other autoimmune diseases are also provided. Thepresent invention further relates to means for detecting, preventing andtreating RA, MS and other autoimmune diseases.

The invention further provides methods of preventing or treating T cellmediated pathologies, including RA and MS, by gene therapy. In thesemethods, pure DNA or RNA encoding for a TCR, an immunogenic fragmentthereof or an anti-idiotype antibody having an internal image of a TCRor an immunogenic fragment is administered to an individual. Vectorscontaining the DNA or RNA and compositions containing such vectors arealso provided for use in these methods.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the variable region sequences of Vβ3, Vβ14 and Vβ17 (SEQ IDNOS. 75, 8 and 7, respectively). The boxed segments depict the CDR1,CDR2 and CDR4 hypervariable regions of each Vβ chain. The sequencesbetween the CDR2 and CDR4 regions represent an overlap between these twohypervariable regions.

FIG. 2(A) shows the location of primers used in polymerase chainreaction amplification of T cell receptor β-chain genes, and 2(B) showsprimer sequences used in polymerase chain reaction (SEQ ID NOS. 47through 55, respectively).

FIG. 3 shows the location and sequence of primers used in polymerasechain reaction amplification of HLA-DR B₁ genes (SEQ ID NOS. 56 through59, respectively). Also shown are HLA-DR allele specificoligonucleotides (SEQ ID NOS. 60 through 71, respectively).

DETAILED DESCRIPTION OF THE INVENTION

The invention generally relates to vaccines and their use forpreventing, ameliorating or treating T cell-mediated pathologies, suchas autoimmune diseases and T cell lymphomas. Vaccination provides aspecific and sustained treatment which avoids problems associated withother potential avenues of therapy.

As used herein, the term “T cell-mediated pathology” refers to anycondition in which an inappropriate T cell response is a component ofthe pathology. The term is intended to encompass both T cell mediatedautoimmune diseases and diseases resulting from unregulated clonal Tcell replication. In addition, the term is intended to include bothdiseases directly mediated by T cells and those, such as myastheniagravis, which are characterized primarily by damage resulting fromantibody binding, and also diseases in which an inappropriate T cellresponse contributes to the production of those antibodies.

As used herein, “substantially the amino acid sequence,” or“substantially the sequence” when referring to an amino acid sequence,means the described sequence or other sequences having any additions,deletions or substitutions that do not substantially effect the abilityof the sequence to elicit an immune response against the desired T cellreceptor sequence. Such sequences commonly have many other sequencesadjacent to the described sequence. A portion or segment of thedescribed immunizing sequence can be used so long as it is sufficientlycharacteristic of the desired T cell receptor or fragment thereof tocause an effective immune response against desired T cell receptors, butnot against undesired T cell receptors. Such variations in the sequencecan easily be made, for example by synthesizing an alternative sequence.The alternate sequence can then be tested, for example by immunizing avertebrate, to determine its effectiveness.

As used herein, the term “fragment” means an immunogenically effectivesubset of the amino acid sequence that comprises a TCR. The term isintended to include such fragments in conjunction with or combined withadditional sequences or moieties, as for example where the peptide iscoupled to other amino acid sequences or to a carrier. The terms“fragment” and “peptide” can, therefore, be used interchangeably since apeptide will be the most common fragment of the T cell receptor. Eachfragment of the invention can have an altered sequence, as describedabove for the term “substantially the sequence.”

Reference herein to a “fragment,” “portion” or “segment” of a T cellreceptor does not mean that the composition must be derived from intactT cell receptors. Such “fragments,” portions” or “segments” can beproduced by various means well-known to those skilled in the art, suchas, for example, manual or automatic peptide synthesis, various methodsof cloning or enzymatic treatment of a whole TCR.

As used herein when referring to the relationship between peptidefragments of the invention and sequences of TCRs, “corresponding to”means that the peptide fragment has an amino acid sequence which issufficiently homologous to a TCR sequence or fragment thereof tostimulate an effective regulatory response in the individual. Thesequence, however, need not be identical to the TCR sequence as shown,for instance, in Examples II and III.

By “immunogenically effective” is meant an amount of the T cell receptoror fragment thereof which effectively elicits an immune response toprevent or treat a T cell mediated pathology or an unregulated T cellclonal replication in an individual. Such amounts will vary betweenspecies and individuals depending on many factors for which one skilledin the art can determine.

As used herein, “binding partner” means a compound which is reactivewith a TCR. Generally, this compound will be a Major HistocompatibilityAntigen (MHC) but can be any compound capable of directly or indirectlystimulating T cell activation or proliferation when bound to the TCR.Such compounds can also, for example, be a superantigen that binds to asuperantigen binding site on the TCR.

As used herein, “individual” means any vertebrate, including humans,capable of having a T cell mediated pathology or unregulated clonal Tcell replication and is used interchangeably with “vertebrate.”

As used herein, “ligand” means any molecule that reacts with anothermolecule to form a complex.

As used herein, “selectively binds” means that a molecule binds to onetype of molecule or related group of molecules, but not substantially toother types of molecules. In relation to Vβs, “selective binding”indicates binding to TCRs or fragments thereof containing a specific Vβwithout substantial cross-reactivity with other TCRs that lack thespecific Vβ.

The immune system is the primary biological defense of the host (self)against potentially pernicious agents (non-self). These perniciousagents may be pathogens, such as bacteria or viruses, as well asmodified self cells, including virus-infected cells, tumor cells orother abnormal cells of the host. Collectively, these targets of theimmune system are referred to as antigens. The recognition of antigen bythe immune system rapidly mobilizes immune mechanisms to destroy thatantigen, thus preserving the sanctity of the host environment.

The principal manifestations of an antigen-specific immune response arehumoral immunity (antibody mediated) and cellular immunity (cellmediated). Each of these immunological mechanisms are initiated throughthe activation of helper (CD4+) T Cells. These CD4+ T cells in turnstimulate B cells, primed for antibody synthesis by antigen binding, toproliferate and secrete antibody. This secreted antibody binds to theantigen and facilitates its destruction by other immune mechanisms.Similarly, CD4+ T cells provide stimulatory signals to cytotoxic (CD8+)T cells that recognize and destroy cellular targets (for example, virusinfected cells of the host). Thus, the activation of CD4+ T cells is theproximal event in the stimulation of an immune response. Therefore,elaboration of the mechanisms underlying antigen specific activation ofCD4+ T cells is crucial in any attempt to selectively modifyimmunological function.

T cells owe their antigen specificity to the T cell receptor (TCR) whichis expressed on the cell surface. The TCR is a heterodimericglycoprotein, composed of two polypeptide chains, each with a molecularweight of approximately 45 kD. Two forms of the TCR have beenidentified. One is composed of an alpha chain and a beta chain, whilethe second consists of a gamma chain and a delta chain. Each of thesefour TCR polypeptide chains is encoded by a distinct genetic locuscontaining multiple discontinuous gene segments. These include variable(V) region gene segments, joining (J) region gene segments and constant(C) region gene segments. Beta and delta chains contain an additionalelement termed the diversity (D) gene segment. Since D segments andelements are found in only some of the TCR genetic loci, andpolypeptides, further references herein to D segments and elements willbe in parentheses to indicate the inclusion of these regions only in theappropriate TCR chains. Thus, V(D)J refers either to VDJ sequences ofchains which have a D region or refers to VJ sequences of chains lackingD regions.

With respect to the beta chain of the variable region referred to as aVβ, the nomenclature used herein to identify specific Vβs follows thatof Kimura et al., Eur. J. Immuno. 17:375-383 (1987), with the exceptionthat the Vβ14 herein corresponds to Vβ3.3 of Kimura et al.

During lymphocyte maturation, single V, (D) and J gene segments arerearranged to form a functional gene that determines the amino acidsequence of the TCR expressed by that cell. Since the pool of V, (D) andJ genes which may be rearranged is multi-membered and since individualmembers of these pools may be rearranged in virtually any combination,the complete TCR repertoire is highly diverse and capable ofspecifically recognizing and binding the vast array of binding partnersto which an organism may be exposed. However, a particular T cell willhave only one TCR molecule and that TCR molecule, to a large degree ifnot singly, determines the specificity of that T cell for its bindingpartner.

Animal models have contributed significantly to the understanding of theimmunological mechanisms of autoimmune disease. One such animal model,experimental allergic encephalomyelitis (EAE), is an autoimmune diseaseof the central nervous system that can be induced in mice and rats byimmunization with myelin basic protein (MBP). The disease ischaracterized clinically by paralysis and mild wasting andhistologically by a perivascular mononuclear cell infiltration of thecentral nervous system parenchyma. The disease pathogenesis is mediatedby T cells having specificity for MBP. Multiple clones of MBP-specific Tcells have been isolated from animals suffering from EAE and have beenpropagated in continuous culture. After in vitro stimulation with MBP,these T cell clones rapidly induce EAE when adoptively transferred tohealthy hosts. Importantly, these EAE-inducing T cells are specific notonly for the same antigen (MBP), but usually also for a single epitopeon that antigen. These observations indicate that discrete populationsof autoaggressive T cells are responsible for the pathogenesis of EAE.

Analysis of the TCRs of EAE-inducing T cells has revealed restrictedheterogeneity in the structure of these disease-associated receptors. Inone analysis of 33 MBP-reactive T cells, only two alpha chain V regiongene segments and a single alpha chain J region gene segment were found.Similar restriction of beta chain TCR gene usage was also observed inthis T cell population. Only two beta chain V region segments and two Jregion gene segments were found. More importantly, approximately eightypercent of the T cell clones had identical amino acid sequences acrossthe region of beta chain V-D-J joining. These findings confirm thenotion of common TCR structure among T cells with similar antigenspecificities and indicate that the TCR is an effective target forimmunotherapeutic strategies aimed at eliminating the pathogenesis ofEAE.

An alternative mechanism for T cell activation has been suggested inwhich endogenous and exogenous superantigens have been shown to mediateT-cell stimulation as described, for example, in White et al, Cell56:27-35 (1989) and Janeway, Cell 63:659-661 (1990).

As used herein, “superantigens” means antigens or fragments thereof thatbind preferentially to T cells at specific sites on the β chain of a TCRand stimulate T cells at very high frequency rate. Such superantigenscan be endogenous or exogenous. “Frequency” refers to the proportion ofT cells responding to antigens and ranges from about ⅕ to {fraction(1/100)} in response to superantigens. Thus, superantigens aredistinguishable from conventional antigens, which have a much lower Tcell response frequency rate ranging from about 1/10⁴ to 1/10⁶.Superantigens activate T cells by binding to specific Vβs. Thesuperantigen binding sites of various TCRs have been distinguished fromthe conventional hypervariable regions (CDRs) of TCRs. These CDRsrepresent the regions of TCRs thought to be responsible for bindingconventional antigens that are completed to MHC.

The present invention provides an effective method of immunotherapy forT cell mediated pathologies, including autoimmune diseases, which avoidsmany of the problems associated with previously suggested methods oftreatment. By vaccinating, rather than passively administeringheterologous antibodies, the host's own immune system is mobilized tosuppress the autoaggressive T cells. Thus, the suppression is persistentand may involve any or all immunological mechanisms in effecting thatsuppression. This multi-faceted response is more effective than theuni-dimensional suppression achieved by passive administration ofmonoclonal antibodies or ex vivo-derived regulatory T cell clones whichrequires a highly individualized therapeutic approach because of MHCnon-identity among humans in order to avoid graft versus host reactions.The methods of the present invention are also more effective thanvaccination with attenuated disease-inducing T cells that lackspecificity for the protective antigen on the surface of a particular Tcell as well as the variable induction of immunity to that antigen. Inaddition, vaccination with attenuated T cells is plagued by the samelabor intensiveness and need for individualized therapies as noted abovefor ex vivo derived regulatory T cell clones.

As they relate to autoimmune disease, the vaccine peptides of thepresent invention comprise TCRs or immunogenic fragments thereof fromspecific T cells that mediate autoimmune diseases. The vaccines can bewhole TCRs substantially purified from T cell clones, individual T cellreceptor chains (for example, alpha, beta, etc.) or portions of suchchains, either alone or in combination. The vaccine can be homogenous,for example, a single peptide, or can be composed of more than one typeof peptide, each of which corresponds to a different portion of the TCR.Further, these peptides can be from different TCRs that contribute tothe T cell mediated pathology. These vaccine peptides can be of variablelength so long as they can elicit a regulatory response. Preferably,such peptides are between about 5-100 amino acids in length, and morepreferably between about 6-25 amino acids in length.

In a specific embodiment, the immunizing peptide can have the amino acidsequence of a β-chain VDJ region when the subject has MS or RA. Anyimmunogenic portion of these peptides can be effective, particularly aportion having substantially the sequence SGDQGGNE (SEQ ID No. 1) orCAIGSNTE (SEQ ID No. 2) of Vβ4 and Vβ12, respectively, for MS; or,substantially the sequence ASSLGGAVSYN (SEQ ID No. 3), ASSLGGEETQYF (SEQID No. 4), ASSLGGFETQYF (SEQ ID No. 5) or ASSLGGTEAFF (SEQ ID No. 6) forRA. Thus, amino acid substitutions can be made which do not destroy theimmunogenicity of the peptide. Additionally, this peptide can be linkedto a carrier to further increase its immunogenicity. Alternatively,whole T cell receptors or TCR fragments that include these sequences canbe used to vaccinate directly.

In a further specific embodiment, T cell receptors, whole T cells orfragments of TCRs that contain Vβ17, Vβ14 or Vβ3 can be used to immunizean individual having a T cell mediated pathology to treat or prevent thedisease. In a specific embodiment, rheumatoid arthritis can be sotreated. The immune response generated in the individual can neutralizeor kill T cells having Vβ17, Vβ14 or Vβ3 and, thus, prevent or treat thedeleterious effects of such Vβ-bearing T cells. Moreover, to the extentthat Vβ17, Vβ14 or Vβ3 is common to T cell receptors on pathogenic Tcells mediating other autoimmune diseases or autoimmune diseases ingeneral, such vaccines can also be effective in ameliorating such otherautoimmune diseases.

As used herein, “Vβ17” refers to a specific human β-chain variableregion of three T cell receptors. Vβ17 has the following amino acidsequence: MSNQVLCCVVLCFLGANTVDGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIVNDFQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASS (SEQ ID No. 7).

“Vβ14” refers to a specific human β-chain variable regions of anotherTCR. Vβ14 has the following amino acid sequence:MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASS (SEQ ID No. 8).

“Vβ3” refers to a family of specific human β-chain variable region. Twomembers of the Vβ3 family have been identified as Vβ3.1 and Vβ3.2. Vβ3.1has the following amino acid sequence: MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASS (SEQ ID No. 9). Vβ3.2 has thefollowing amino acid sequence: MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVDMDHENMFWYQRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASS (SEQ ID No. 10).

The hypervariable or junctional regions are useful for the vaccines ofthe present invention. Hypervariable regions useful in the presentinvention include CDR1, CDR2, CDR3 and CDR4. The amino acid sequences ofthe CDR1, CDR2 and CDR4 hypervariable regions for Vβ3, Vβ14 and Vβ17 areshown in FIG. 1.

The CDR3, also known as the V(D)J region, is useful as a vaccine of thepresent invention since T cell immunity elicited by peptidescorresponding to this region is expected to be highly specific for aparticular antigen. Due to the recombination of the V, D and J regiongenes prior to maturation, the amino acid sequence across these regionsis virtually unique to each T cell and its clones.

However, as a germ-line element, the CDR2 region is also useful in humandiseases such as MS and in particular RA. In RA studies, the resultsindicate a limited number of Vβs among the activated T cellsinfiltrating the synovial target tissue with only a few incidences ofsequence homology in the βVDJ region. Thus, peptides corresponding tothe CDR2 region are viable alternatives for use as vaccines of thepresent invention. For example, the CDR2 region of Vβ3,DPGLGLRLIYFSYDVKMKEKG (SEQ ID No.72), of Vβ14, DPGLGLRQIYYSMNVEVTDKG(SEQ ID No.73), or of Vβ17, DPGQGLRLIYYSQIVNKFQKG (SEQ ID No.74), can beused.

Modifications in these sequences that do not affect the ability of thereceptor or an immunogenic fragment thereof to act as an immunogen tostimulate the desired immune response are contemplated and are includedin the definition of TCR fragment. The variable region can be joinedwith any D and J segment of the TCR. Further, immunogenicallyrepresentative fragments of Vβ3, Vβ14 and Vβ17 are also included in thedefinition of “Vβ3,” “Vβ14” and “Vβ17,” respectively.

By “substantially pure,” it is meant that the TCR is substantially freeof other biochemical moieties with which it is normally associated innature. Such substantially pure TCRs or fragments thereof, for instance,can be synthesized, produced recombinantly by means known to thoseskilled in the art. In addition, whole TCRs can be enzymatically treatedto produce such fragments.

In another embodiment, vaccine peptides can correspond to the Vβ regionsthat contain sequences of high homology which are conserved amongpathogenic TCRs. These regions of conserved homology include theconventional CDRs, such as CDR1 and CDR2, which are common to T cellsbearing the same Vβ, and also the superantigen binding site, which canbe common to pathogenic TCRs bearing different Vβs. The superantigenbinding site is also known to be in or around the CDR4 hypervariableregion.

The vaccines of the present invention comprise peptides of varyinglengths corresponding to the TCR or immunogenic fragments thereof. Thevaccine peptides can correspond to regions of the TCR which distinguishthat TCR from other nonpathogenic TCRs. Such specific regions can, forexample, be located within the various region(s) of the respective TCRpolypeptide chains, for example, a short sequence spanning the V(D)Jjunction, thus restricting the immune response solely to those T cellsbearing this single determinant.

The vaccines are administered to a host exhibiting or at risk ofexhibiting an autoimmune response. Definite clinical diagnosis of aparticular autoimmune disease warrants the administration of therelevant disease-specific TCR vaccines. Prophylactic applications arewarranted in diseases where the autoimmune mechanisms precede the onsetof overt clinical disease (for example, Type I Diabetes). Thus,individuals with familial history of disease and predicted to be at riskby reliable prognostic indicators could be treated prophylactically tointerdict autoimmune mechanisms prior to their onset.

TCR peptides can be administered in many possible formulations,including pharmaceutically acceptable mediums. In the case of a shortpeptide, the peptide can be conjugated to a carrier, such as KLH, inorder to increase its immunogenicity. The vaccine can include or beadministered in conjunction with an adjuvant, of which several are knownto those skilled in the art. After initial immunization with thevaccine, further boosters can be provided. The vaccines are administeredby conventional methods, in dosages which are sufficient to elicit animmunological response. Such dosages can be easily determined by thoseskilled in the art.

Appropriate peptides to be used for immunization can be determined asfollows. Disease-inducing T cell clones reactive with the targetantigens are isolated from affected individuals. Such T cells areobtained preferably from the site of active autoaggressive activity suchas a lesion in the case of pemphigus vulgaris, the central nervoussystem (CNS) in the case of multiple sclerosis or the synovial fluid ortissue in the case of rheumatoid arthritis. Alternatively, such T cellscan be obtained from blood of affected individuals. The TCR genes fromthese autoaggressive T cells are then sequenced. Polypeptidescorresponding to TCRs or portions thereof that are selectivelyrepresented among disease inducing T cells (relative to non-pathogenic Tcells) can then be selected as vaccines and made and used as describedabove. An alternative method for isolating pathogenic T cells isprovided by Albertini in PCT Publication No. WO88/10314, published onDec. 29, 1988.

Alternatively, the vaccines can comprise anti-idiotypic antibodies whichare internal images of the peptides described above. Methods of making,selecting and administering such anti-idiotype vaccines are well knownin the art. See, for example, Eichmann, et al., CRC Critical Reviews inImmunology 7:193-227 (1987), which is incorporated herein by reference.

In a further aspect of the present invention, methods of preventing theproliferation of T cells associated with a T cell mediated pathology arealso contemplated. Such methods include determining a T cell receptorbinding partner according to the above methods and administering aneffective amount of such binding partner in an appropriate form toprevent the proliferation of the T cells. The methods can be used, forexample, to build a tolerance to self antigens as in the case of anautoimmune disease.

The present invention also relates to other methods of preventing ortreating a T cell pathology by inhibiting the binding of a T cellreceptor to its TCR binding partner in order to prevent theproliferation of T cells associated with the T cell pathology. Ligandsthat are reactive with the T cell receptor or its binding partner atbinding sites that inhibit the T cell receptor attachment to the bindingpartner can be used. Such ligands can be, for example, antibodies havingspecificity for the T cell receptor or its binding partner.

The invention also provides a method of preventing or treating a T cellmediated pathology in an individual comprising cytotoxically orcytostatically treating a Vβ-containing T-cells, particularly Vβ3, Vβ14and Vβ17, in the individual. The Vβ-containing T cells are treated witha cytotoxic or cytostatic agent that selectively binds to the Vβ regionof a T cell receptor that mediates a pathology, such as RA or MS forexample. The agent can be an antibody attached to a radioactive orchemotherapeutic moiety. Such attachment and effective agents are wellknown in the art. See, for example, Harlow, E. and Lane, Antibodies, ALaboratory Manual, Cold Spring Harbor Laboratory (1988), which isincorporated herein by reference.

Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a T cell mediated autoimmune disease. Theinvention describes clonal infiltrates of activated Vβ3, Vβ14 and Vβ17 Tcells in the synovium of rheumatoid arthritis patients. The presence ofthese T cells in the diseased tissue of most of patients examined, theirclonality, and the cytotoxic activity of one such T cell for synovialadherent cells, demonstrate a central role for T cells bearing these Vβsin the pathogenesis of RA.

Activated T cell populations in the synovial tissue of RA patients havebeen examined by analyzing T cell receptor (TCR) mRNAs isolated fromIL-2 receptor positive (IL-2R+) synovial T cells. As described inExample X(C), TCR mRNAs were amplified using a polymerase chain reaction(PCR) protocol designed to amplify human TCR β-chain genes containingvirtually any desired Vβ gene element. In this analysis, clonal Vβ17rearrangements were found to be enriched in the IL2-R+population,indicating that Vβ17 T cells are likely involved in the pathogenesis ofRA. A CD4+, Vβ17 bearing T cell clone has been isolated from one of thesynovial tissue specimens and its in vitro cytotoxicity for synovialadherent cells supports the direct involvement of Vβ17 T cells in RA.

Additional studies were conducted to ensure that the prevalence of Vβ17T cells in the initial studies did not result from an amplification biasfor the Vβ consensus primer, and to examine the involvement of other TCRVβ gene families in RA. As described in Example XI, RNAs from activated(IL2-R+) synovial T cells were analyzed by PCR-amplification with apanel of Vβ-specific PCR primers. In this analysis, Vβ17 transcriptswere found in four of the five patients tested, confirming theassociation of Vβ17 with RA and validating the utility of the Vβconsensus primer. In addition, Vβ14 was found in four of the five RApatient samples and Vβ3 and Vβ9 were detectable in three of fivepatients.

The sequences of these various Vβ polypeptides were examined forhomology to Vβ17. The results are reported in Table 1.

TABLE 1 Relative Homologies of TCR β Chain Polypeptides With Human Vβ17hVβ  100% (94aa) mVβ6 69.1% (94aa) hVβ3 58.5% (94aa) hVβ12.1 53.2%(94aa) hVβ14 52.1% (94aa) mVβ7 51.7% (89aa) hVβ9 33.0% (94aa) hVβ =human Vβ mVβ = mouse Vβ

As shown in Table 1, mouse Vβ6 is most closely homologous, followed byhVβ3, hVβ12.1, hVβ14, and mVβ7. Three of the human Vβs, Vβ3, Vβ14 andVβ17, were detected in the synovium of RA patients. Vβ12.1 wasnegligible in the synovium despite considerable overall homology withVβ17. In contrast, Vβ9 was found in three of five synovial samples, yetis only weakly homologous to Vβ17.

A surprising discovery is the greater homology found among all of theVβs detected in the synovia of RA patients, except Vβ9, in a contiguousstretch of 15 amino acids located carboxy to the CDR2 region. The 15amino acid sequences of these Vβs as well as other human and mouse Vβsare shown in Table 2. Within the β-chain, this region of conservationcorresponds positionally to that previously shown to containsuperantigen binding sites.

TABLE Proposed Superantigen Binding Site in RA Associated Vβ Genes %H¹Seq. Id. No.       +- hVβ17 EGYSVSREKKESFPL 11       +- hVβ3EGYSVSREKKERFSL  86.7 12        +- hVβ14 EGYKVSRKEKRNFPL  66.7 13hVβ12.1 DGYSVSRSKTEDFLL  66.7 14 hVβ9 NRFSPKSPDKAHLNL <30 15       +-mVβ6 EGYDASREKKSSFSL  73.3 16          +- mVβ7 KGYRVSRKKREHFSL  64.3 17mVβ8.2a DGYKASRPSQENFSL 18           +- mVβ8.2c ..........KE... 19hVβ13.2 DGYNVSRLKKQNFLLGLE 20 %H = % homology compared with Vβ17

The exogenous superantigen, SEC₂, stimulates human Vβ13.2 T cells as aresult of binding to a site on Vβ13.2 as described in Choi et al.,Nature 346:471-473 (1990), which is incorporated herein by reference.The sequence of this binding site is shown in Table 2 as the last 11amino acids for Vβ13.2.

A binding site for Mls-1a, an endogenous superantigen, has also beenmapped to this region as described in Pullen et al., Cell 61:1365-1374(1990), which is incorporated herein by reference. Identification ofthis region as the Mls binding site involved the study of Vβ8.2a, theVβ8.2 isoform common to laboratory mice, and Vβ8.2c, a β-chain found inwild mice. These β-chain polypeptides are distinguished functionally bytheir differential reactivities with Mls-1a and structurally by adifference of five amino acids. Of particular importance are theresidues at position 70 and 71. The responder β-chain, Vβ8.2c, haslysine and glutamic acid, respectively, at these positions. Specificmutagenesis of the non-responder gene to encode a lysine-glutamic acidpair at positions 70 and 71 rendered that non-responder β-chainMls-1a-reactive. This confirms the region as one of superantigenbinding. Thus, both these exogenous and endogenous superantigens canbind in the vicinity of the 15 amino acid sequence homology identifiedin Table 2. In addition, the lysine-glutamic acid pair charge motif isimplicated in Mls-1a reactivity. Involvement of this charge motif inMls-1a reactivity is confirmed by its presence in mouse Vβ6 and Vβ7, twoother Mls-1a reactive murine β-chains. The Vβ8.2c, Vβ6 and Vβ7 chargemotif of a lysine or arginine followed by a glutamic acid residue isunderlined in Table 2. Thus, the superantigen binding site for Mls-1a ischaracterized by this charge motif contained within the region of localhomology.

The present invention is directed to the unexpected discovery that humanVβ3, Vβ14 and Vβ17 have a region that corresponds to the Mls-1a bindingsite. These three human Vβs display a significant degree of overallhomology within the entire 94 amino acid sequence and of local homologywithin the 15 amino acid sequence with mVβ6 and mVβ7. Each of these Vβspossess a lysine or arginine/glutamic acid pair, which are underlined inTable 2 and represent what is meant by the term “charge motif.” Vβ12.1,while displaying a high degree of overall and local homology with Vβ3,Vβ14 and Vβ17, lacks the charge motif, perhaps accounting for itspresence in the synovium of only one of five RA patients. Vβ9 shows nooverall or local homology to Vβs 3, 14 or 17 and lacks the charge motif.

The presence of Vβ3, Vβ14 and Vβ17-bearing T cells has been demonstratedamong the activated synovial T cells in RA. These three β-chainpolypeptides, in contrast to other known Vβs, possess overall and localsequence homology and an apparent superantigen binding charge motif.These results indicate that Vβ-specific T cell activation bysuperantigen plays a role in RA.

Vβ3, Vβ14 and Vβ17 are the only known human Vβ chains to possess thisapparent superantigen binding site characterized by this local sequencehomology and the identified charge motif. However, it is possible thatother Vβs may become known that contain such binding sites. Thus, asubstantially pure Vβ3, Vβ14 or Vβ17 sequence containing the chargemotif can be used as an immunogen in the vaccines of the presentinvention. For example, the sequences or fragments thereof shown inTable 2 for Vβ3, Vβ14 and Vβ17 can be used. Vaccines containing anycombination of these three Vβ sequences, including all three sequences,can be used effectively to ameliorate T cell associated diseases.

In addition, other common V(D)J sequences of the β-chain observed in RApatients are listed in Table 3. The results taken from two different RAstudies show sequence homologies in the βVDJs from four differentclones, which indicates the usefulness of peptides corresponding to theCDR3 region as appropriate vaccine candidates.

TABLE 3 β-Chain VDJ Seqruences Found in Common in RA Patients Patient VβVDJ Seauence Jβ Seq. ID No. 1012 Vβ14 A S S L G G A V S — Y N Jβ2.1 31013 Vβ3 A S S L G G E E T Q Y F Jβ2.5 4 C Vβ3 A S S L G G F E T Q Y FJβ2.5 5 A Vβ3 A S S L G G T E A — F F Jβ1.1 6

As noted, the invention provides the discovery that specific variableregions of the β-chains of three TCRs, designated Vβ3, Vβ14, and Vβ17,are closely associated with T cell mediated pathologies, especiallyrheumatoid arthritis in human subjects. This discovery allows for thedetection, prevention and treatment of rheumatoid arthritis using themethodology set out in this invention. Similar therapeutic approachesset out above for EAE can be applied to rheumatoid arthritis by thoseskilled in the art.

Specifically, the invention provides a method of diagnosing orpredicting susceptibility to T cell mediated pathologies in anindividual comprising detecting T cells having the β-chain variableregion from Vβ3, Vβ14 or Vβ17 in a sample from the individual, thepresence of abnormal levels of such Vβ-containing T cells indicating thepathology or susceptibility to the pathology. The Vβ-containing T cellscan be qualitatively or quantitatively compared to that of normalindividuals. Such diagnosis can be performed, for example, by detectinga portion of the Vβs that does not occur on non-rheumatoid arthritisassociated β-chain variable region T-cell receptors. The Vβs of thepresent invention can be detected, for example, by contacting the Vβswith a detectable ligand capable of specifically binding to theindividual Vβs. Many such detectable ligands are known in the art, e.g.an enzyme linked antibody. Alternatively, nucleotide probes,complementary to the individual Vβ of interest, encoding nucleic acidsequences can be utilized to detect such Vβ-containing T cells, astaught, for instance, in Examples X and XI.

The invention also provides a method of preventing or treating a T cellmediated pathology comprising preventing the attachment of a Vβ3-, Vβ14-or Vβ17-containing T-cell receptor to its binding partner. In oneembodiment, attachment is prevented by binding a ligand to Vβ3, Vβ14 orVβ17. In an alternative embodiment, attachment is prevented by binding aligand to the Vβ3, Vβ14 or Vβ17 binding partner. Attachment can beprevented by known methods, e.g. binding an antibody to the individualVβs or to its binding partner in order to physically block attachment.

Multiple Sclerosis

T cells causative of multiple sclerosis (MS) have not previously beenidentified, though MBP-reactive T cells have been proposed to play arole due to the clinical and histologic similarities between MS and EAE.In rat and mouse models of EAE, MBP-reactive, encephalitogenic T cellsshow striking conservation of β-chain V(D)J amino acid sequence, despiteknown differences in MHC restriction and MBP-peptide antigenspecificity. One embodiment of the invention is premised on theobservation that a human myelin basic protein (MBP)-reactive T cellline, derived from an MS patient, has a TCR β-chain with a V(D)J aminoacid sequence of Vβ4 homologous with that of β-chains from MBP-reactiveT cells mediating pathogenesis in experimental allergicencephalomyelitis (EAE), an animal model of MS, as shown in Table 4.This finding demonstrates the involvement of MBP-reactive T cells in thepathogenesis of MS and demonstrates that TCR peptides similar to thosedescribed herein for the prevention of EAE can be appropriate intreating MS. As shown in Table 4, a VDJ sequence of Vβ12, CAIGSNTE (SEQID No. 2) and one of Vβ4, SGDQGGNE (SEQ ID No. 1), have been observed inMS patients.

TABLE 4 β-Chain VDJ Sequences in MS Patients Homologous to Rat VDJSequences Vβ VDJ Sequence SEQ ID Nos. RAT Vβ8.2 S S D S S N T E 21 RATVβ8.2 S S D S G N T E 22 HUMAN Vβ4 S G D Q G G N E 1 HUMAN Vβ12 C A I GS N T E 2

The activated cells of one MS patient from the CSF were analyzed andfound to be predominantly Vβ14 and Vβ3. In both cases, there was apredominate clone of Vβ14 and a predominate clone of Vβ3. These findingsindicate that the results from the RA studies relating to the same Vβscan be extended to other autoimmune pathologies, including MS.

In this regard, the invention is directed to the discovery that β-chainVDJ fragments homologous to VDJ sequences found in rodent EAE, such asSGDQGGNE (SEQ ID No.1) and CAIGSNTE (SEQ ID No. 2), are closelyassociated with multiple sclerosis in human subjects. This discoveryallows for the detection, prevention and treatment of multiple sclerosisusing the methodology set out in this invention. Similar therapeuticapproaches set out herein for EAE can be applied to multiple sclerosisby those skilled in the art.

Specifically, the invention provides a method of diagnosing orpredicting susceptibility to multiple sclerosis in an individualcomprising detecting T cells having various Vβs, such as Vβ4 or Vβ12,and particularly having substantially the sequence SGDQGGNE (SEQ IDNo.1) or CAIGSNTE (SEQ ID No.2), in a sample from the individual, thepresence of the sequence indicating multiple sclerosis or susceptibilityto multiple sclerosis. The sequences can be detected, for example, bycontacting T cells or TCRs with a detectable ligand. Many such ligandsare known in the art, for example, an enzyme linked or otherwise labeledantibody specific for the sequence. Alternatively, nucleotide probescomplementary to the nucleic acid encoding the sequence can be utilizedas taught, for instance, in Example IX.

The invention also provides a method of preventing or treating multiplesclerosis comprising preventing the attachment of a T-cell receptorcontaining various Vβs, including Vβ4, Vβ12 or fragments thereof, suchas those having substantially the SGDQGGNE (SEQ ID No.1) or CAIGSNTE(SEQ ID No.2) sequence to its binding partner. In one embodiment,attachment is prevented by binding a ligand to the sequence. In analternative embodiment, attachment is prevented by binding a ligand tothe binding partner. Attachment can be prevented by known methods, suchas binding an antibody to these Vβs, and in particular to the SGDQGGNE(SEQ ID No.1) or CAIGSNTE (SEQ ID No.2) sequences, to physically blockattachment.

The invention also provides a method of preventing or treating multiplesclerosis in an individual comprising cytotoxically or cytostaticallytreating T cells containing various Vβs, including Vβ4, Vβ12 andfragments thereof, particularly those having substantially the SGDQGGNE(SEQ ID No.1) or CAIGSNTE (SEQ ID No.2) sequence in the individual. Inone embodiment, T-cells are treated with a cytotoxic or cytostatic agentwhich selectively binds to these Vβs or their immunogenic fragments. Theagent can be, for example, an antibody attached to a radioactive orchemotherapeutic moiety.

T Cell Pathologies of Malignant Etiology

To illustrate the utility of TCR vaccination, autoimmune disease hasbeen discussed. However, T cell lymphoma is another T cell pathologywhich would be amenable to this type of treatment. Application of thistechnology in the treatment of T lymphoma would be conducted invirtually identical fashion. In one respect, however, this technology ismore readily applied to T cell proliferative disease since the isolationof the pathogenic T cells is more easily accomplished. Once the clonesare isolated, the technology is applied in the manner described herein.Specifically, the TCR genes of the T lymphomas are sequenced,appropriate regions of those TCRs are identified and used as vaccines.The vaccines can comprise single or multiple peptides, and can beadministered in pharmaceutically acceptable formulations, with orwithout adjuvants, by conventional means.

Gene Therapy

The present invention further relates to an alternative method oftreating or preventing a T cell mediated pathology by gene therapy. Inthis method, a nucleic acid encoding for a TCR or an immunogenicfragment thereof is first inserted into an appropriate delivery system,for example a plasmid. The nucleic acid can be DNA or RNA encoding forTCRs, immunogenic fragments thereof or anti-idiotype antibodies that canbe used as vaccines in the present invention. Such DNA or RNA can beisolated by standard methods known in the art. The isolated nucleic acidcan then be inserted into a suitable vector by known methods. Suchmethods are described, for example, in Maniatis et al., MolecularCloning: A Laboratory Manual (Cold Spring Harbor Laboratory 1982), whichis incorporated herein by reference.

The vector is subsequently administered directly into a tissue of anindividual. Preferably, the DNA or RNA-containing vector is injectedinto the skeletal muscle of the individual. For example, a 1.5 cmincision can be made to expose the quadricep muscles of the subject. A0.1 ml solution containing from 10-100 μg of a DNA or RNA plasmid and5-20% sucrose is injected over 1 minute into the exposed quadricepmuscles about 0.2 cm deep. The skin is thereafter closed. The amount ofDNA plasmid or RNA can range from 10 to 100 μl of hypotonic, isotonic orhypertonic sucrose solutions or sucrose solutions containing 2 mM CaCl₃.The plasmid containing solutions can also be administered over a longerperiod of time, for example, 20 minutes, by infusion. The in vivoexpression of the desired gene can be tested by determining an increasedproduction of the encoded polypeptide by the subject according tomethods known in the art or as described, for example, in Wolff et al.,Science 247:1465-1468 (1990).

It is believed that the treated cells will respond to the directinjection of DNA or RNA by expressing the encoded polypeptide for atleast about 60 days. Thus, the desired TCR, immunogenic fragment oranti-idiotype antibody can be effectively expressed by the cells of theindividual as an alternative to vaccinating with such polypeptides.

The present invention also relates to vectors useful in the gene therapymethods and can be prepared by methods known in the art. Compositionscontaining such vectors and a pharmaceutically acceptable medium arealso provided. The pharmaceutically acceptable medium should not containelements that would degrade the desired nucleic acids.

The following examples are intended to illustrate but not limit theinvention.

EXAMPLE I Rat Model of EAE

Female Lewis rats, (Charles River Laboratories, Raleigh-Durham, NC) wereimmunized in each hind foot pad with 50 μg of guinea pig myelin basicprotein emulsified in complete Freund's adjuvant. The first signs ofdisease were typically observed 9-11 days post-immunization. Diseaseseverity is scored on a three point scale as follows: 1=limp tail;2=hind leg weakness; 3=hind leg paralysis. Following a disease course ofapproximately four to six days, most rats spontaneously recovered andwere refractory to subsequent EAE induction.

EXAMPLE II Selection and Preparation of Vaccines

Vaccinations were conducted with a T cell receptor peptide whosesequence was deduced from the DNA sequence of a T cell receptor betagene predominating among EAE-inducing T cells of B10.PL mice. The DNAsequence was that reported by Urban, et al., supra, which isincorporated herein by reference. A nine amino acid peptide, having thesequence of the VDJ junction of the TCR beta chain of the mouse, wassynthesized by methods known to those skilled in the art. The sequenceof this peptide is: SGDAGGGYE (SEQ ID No.23). (Amino acids arerepresented by the conventional single letter codes.) The equivalentsequence in the rat has been reported to be: SSDSSNTE (SEQ ID No. 24)(Burns et al., J. Exp. Med. 169:27-39 (1989)). The peptide was desaltedby Sephadex G-25 (Pharmacia Fine Chemicals, Piscataway, N.J.) columnchromatography in 0.1 M acetic acid and the solvent was subsequentlyremoved by two cycles of lyophilization. A portion of the peptide wasconjugated to keyhole limpet hemocyanin (KLH) with glutaraldehyde at aratio of 7.5 mgs of peptide per mg of KLH. The resulting conjugate wasdialyzed against phosphate buffered saline (PBS).

EXAMPLE III Vaccination Against EAE

Vaccines used in these studies consisted of free VDJ peptide and also ofVDJ peptide conjugated to KLH. These were dissolved in PBS and wereemulsified with equal volumes of either (1) incomplete Freund's adjuvant(IFA) or (2) complete Freund's adjuvant (CFA) made by suspending 10mg/ml heat killed desiccated Mycobacterium tuberculosis H37ra (DifcoLaboratories, Detroit, Mich.) in IFA. Emulsions were administered to8-12 week old female Lewis rats in a final volume of 100 microliters peranimal (50 μl in each of the hind footpads). 5 μg of unconjugated VDJpeptide were administered per rat. KLH-VDJ conjugate was administered ata dose equivalent to 10 μg of KLH per rat. Twenty-nine days later eachrat was challenged with 50 μg of guinea pig myelin basic protein incomplete Freund's adjuvant in the front footpads. Animals were monitoreddaily beginning at day 9 for clinical signs of EAE and were scored asdescribed above. The results are presented in Table 5. As can be seen,not only was there a reduced incidence of the disease in the vaccinatedindividuals, but in those which did contract the disease, the severityof the disease was reduced and/or the onset was delayed. The extent ofprotection varied with the vaccine formulation, those including CFA asthe adjuvant demonstrating the greatest degree of protection.

TABLE 5 Animal Vaccination Days After Challenge No. (Adjuvant) 10 11 1213 14 15 16 17 18 1 VDJ (IFA) — — 2 3 3 3 — — — 2 VDJ (IFA) — — 1 3 3 32 — — 3 VDJ (IFA) — — — 3 3 3 2 — — 4 VDJ (CFA) — — — — 1 1 1 — — 5 VDJ(CFA) — — — — — — — — — 6 VDJ (CFA) — — — 1 3 3 3 2 — 7 KLH-VDJ (CFA) —— — 1 3 2 — — — 8 KLH-VDJ (CFA) — — — — 1 1 1 1 — 9 KLH-VDJ (CFA) — — —— — — — — — 10 KLH-VDJ (IFA) — 1 3 3 2 2 1 — — 11 KLH-VDJ (IFA) — — 3 33 3 3 2 — 12 KLH-VDJ (IFA) — — 1 3 3 3 3 — — 13 NONE 1 3 3 3 3 1 — — —14 NONE — 1 3 3 3 1 — — — 15 NONE 1 3 3 3 1 — — — — Scoring: — nosigns 1) limp tail 2) hind leg weakness 3) hind leg paralysis

EXAMPLE IV Vaccination Against EAE with Lewis Rat VDJ Peptides

The VDJ peptide used in the previous examples was synthesized accordingto the sequence of TCR β chain molecules found on EAE-inducing T cellsin B10.PL mice. In addition, peptides were synthesized and tested whichcorrespond to sequences found on encephalitogenic T cells in Lewis rats.These VDJ sequences are homologous with that of B10.PL mice, but notidentical. The rat peptides were synthesized according to the DNAsequences reported by Burns, et al. and Chluba, et al., Eur. J. Immunol.19:279-284 (1989). The sequences of these peptides designated IR1, 2, 3and 9b are shown below, aligned with the B10.PL mouse sequence used inExamples I through III

SEQ ID No. VDJ   S G D A G G Y E 23 IR1 C A S S D — S S N T E V F F G K25 1R2 C A S S D — S G N T E V F F G K 26 1R3 C A S S D — S G N — V L YF G E G S R 27 IR9b  A S S D — S S N T E 28

The preparation, administration and evaluation of these vaccines wereconducted as described in Examples I through III with the followingexceptions: 50 μg of the individual VDJ peptides were incorporated intovaccine formulations containing CFA; neither vaccinations in IFA norvaccinations with peptides conjugated to KLH were conducted. Controlanimals were untreated prior to MBP challenge as in Example III or werevaccinated with emulsions of PBS and CFA to assess the protective effectof adjuvant alone. The results are shown in Table 6 below.

TABLE 6 Animal Vaccination Days After Challenge No. (Adjuvant) 10 11 1213 14 15 16 17 18 1 None — 1 2 3 3 2 — — — 2 None 1 3 3 3 2 — — — — 3None — 2 3 3 3 1 — — — 4 PBS-CFA 1 2 3 3 3 — — — — 5 PBS-CFA 1 2 3 3 3 —— — — 6 PBS-CFA — 2 3 3 3 — — — — 7 IR1 (50 μg) — — — 2 1 — — — — 8 IR1(50 μg) — — — — 1 3 — — — 9 IR1 (50 μg) — — — 1 1 1 1 — — 10 IR2 (50 μg)— — 1 3 3 3 — — — 11 IR2 (50 μg) — — — — 2 2 3 3 — 12 IR2 (50 μg) — — —— 1 — — — — 13 IR3 (50 μg) 1 3 3 3 2 — — — — 14 IR3 (50 μg) — — 2 3 3 —— — — 15 IR3 (50 μg) — — — — — — — — — 16 IR9b (50 μg) — — — — — — — — —17 IR9b (50 μg) — — — — — — — — — 18 IR9b (50 μg) — — — — — — — — — 19IR9b (50 μg) — — — — — — — — — Scoring: — no signs 1) limp tail 2) hindleg weakness 3) hind leg paralysis

As shown in Table 6, disease in unvaccinated control animals wasobserved as early as day 10. Disease was characterized by severeparalysis and wasting, persisted for 4 to 6 days and spontaneouslyremitted. PBS-CFA vaccinated rats displayed disease courses virtuallyindistinguishable from those of unvaccinated controls. In contrast,delays in onset were observed in some of the IR1, 2 or 3 vaccinatedanimals and others showed both delayed onset as well as decreasedseverity and/or duration of disease. Overall, however, vaccinations withthe rat VDJ peptides (IR1-3) were slightly less effective than thosewith the mouse VDJ peptide (Example III). Vaccination with IR9b,however, afforded complete protection in all four animals in which itwas tested. Importantly, no histologic lesions characteristic of diseasewere found in any of the four animals vaccinated with IR9b indicatingthat sub-clinical signs of disease were also abrogated.

EXAMPLE V Vaccination with V Region Specific Peptides

A peptide specific for the Vβ8 gene family was tested as a vaccineagainst EAE. Vβ8 is the most common β chain gene family used byencephalitogenic T cells in both rats and mice. A peptide wassynthesized based on a unique DNA sequence found in the Vβ8 gene, andwhich is not found among other rat Vβ genes whose sequences werereported by Morris, et al., Immunogenetics 27:174-179 (1988). Thesequence of this Vβ8 peptide, designated IR7, is:

IR7 DMGHGLRLIHYSYDVNSTEK (SEQ ID No.29)

The efficacy of this Vβ8 peptide was tested in the Lewis rat model ofEAE (Example I) as described in Examples II and III. 50 μg of peptidewere tested in CFA. Vaccinations in IFA or with peptide-KLH conjugateswere not conducted. The results of these studies are shown in Table 7.

TABLE 7 Animal Vaccination Days After challenge No. (Adjuvant) 10 11 1213 14 15 16 17 18 1 IR7 (50 μg) — — 1 2 3 3 3 — — 2 IR7 (50 μg) — — — —1 1 — — — 3 IR7 (50 μg) — — — — — — — — — Scoring: — no signs 1) limptail 2) hind leg weakness 3) hind leg paralysis

EXAMPLE VI Comparison of Vβ8.2 Peptide Lengths

The results of vaccinations conducted with the rat Vβ8 peptide aresimilar to those observed with the mouse and rat IR1, 2 and 3 peptides.Delayed onset as well as decreased severity and duration of disease wasobserved in one animal. One animal was completely protected.

It has been found that a corresponding 21 amino acid sequence of Vβ8.2(residues 39-59) provided less protection than IR7 as shown in Table 8.The 21 amino acid sequence of Vβ8.2 is DMGHGLRLIHYSYDVNSTEKG (SEQ IDNo.30).

TABLE 8 Comparison of Efficacy of Two β-Chain CDR2 Peptides inProtecting from EAE in the Lewis Rats (50) #With Disease Mean Max MeanMean #With Histology Mean histology Mean S.L. Vaccination #TestedSeverity Onset Duration #Tested Score (14 days) None 10/10  2.8 10.5 8.410/10 3.7 — Control 9/10 2.8 11.5 8.1 9/9 2.6 1.0 Peptide/CFA Vβ8.2₂₀CFA 6/10 1.3 11.2 3.8  8/10 1.0 16.4  PBS/CFA 9/10 2.5 12.1 6.0  9/102.6 — Vβ8.2₂₁ CFA 6/6  3.0 12.0 6.7 6/6 3.3 6.3

Each peptide was used at 100 μg doses and dissolved in saline prior tobeing emulsified in an equal volume of complete Freund's adjuvant (CFA).Animals were challenged after 42 days with 50 μg of guinea pig myelinbasic protein in CFA. The CFA contained 10 mg/ml of mycobacteriatuberculosis. Injections and evaluation of clinical signs and histologywere performed as previously described. Other animals (five per group)were immunized with peptides in the same way and their splenocytes wereremoved after 14 days to test for lymphocyte proliferation as describedin Olee et al., J. Neuroimmunol. 21:235-240 (1989). The sequences of the20 amino acid peptide and the 21 amino acid peptide are designated inTable 8 as Vβ8.2₂₀ and Vβ8.2₂₁ respectively.

EXAMPLE VII Vaccination with J Region Peptides

A peptide was synthesized which corresponds to the J α gene segment,TA39, found among both rat and mouse encephalitogenic T cell receptors.The sequence of this peptide, designated IR5, is:

IR5 RFGAGTRLTVK (SEQ ID No.31)

The efficacy of the JαTA39 peptide was tested in the Lewis rat model ofEAE (Example I) as described in Examples II and III. 50 μg of peptidewere tested in CFA. Vaccinations in IFA or with peptide-KLH conjugateswere not conducted. The results of these studies are shown in Table 9.

TABLE 9 Ani- mal Vaccination Days After Challenge No. (Adjuvant) 10 1112 13 14 15 16 17 18 19 20 1 IR5 (50 μg) — — — — — 2 1 1 1 1 — 2 IR5 (50μg) — — — — — — — — — — — 3 IR5 (50 μg) — — — — — — — — — — — Scoring: —no signs 1) limp tail 2) hind leg weakness 3) hind leg paralysis

The results of vaccinations conducted with the rat J α TA39 peptide aremore effective than those observed with the mouse VDJ peptide or the Vβ8peptide. Two of three animals were totally protected and, in the third,disease onset was markedly delayed. Severity was also reduced in thisanimal though disease persisted for a normal course of 5 days.Importantly, the two animals which were completely protected showed nohistologic evidence of T cell infiltration of the CNS. This resultindicates that vaccinating with the J_(α)TA39 very efficiently induces aregulatory response directed at encephalitogenic T cells. Evensub-clinical signs of disease were abrogated.

EXAMPLE VIII Vaccination with Mixtures of TCR Peptides

Vaccinations were conducted with a mixture of TCR peptides. This mixturecontained 50 μg of each of the peptides IR1, 2, 3 and 5 (the three ratVDJ peptides and the rat JαTA39 peptide).

The efficacy of this peptide mixture was tested in the Lewis rat model(Example I) as described in Examples II and III. Peptides were tested inCFA. Vaccinations in IFA or with peptide-KLH conjugates were notconducted. The results of these studies are shown in Table 10.

TABLE 10 Animal Vaccination Days After challenge No. (Adjuvant) 10 11 1213 14 15 16 17 18 4 IR1, 2, 3, 5 — — — — — — — — — 5 (50 μg each) — — —— — — — — — 6 (50 μg each) — — — — — — — — — Scoring: — no signs 1) limptail 2) hind leg weakness 3) hind leg paralysis

The results of vaccinations conducted with the rat JαTA39 and three VDJpeptides were as effective as those described for IR9b in Table 6. Allthree animals were totally protected. In addition to the absence of anyclinical signs of EAE, two of these three animals were completely freeof histological evidence of T cell infiltration into the CNS while thethird showed only two small foci of lymphocytic infiltration at the baseof the spinal cord.

EXAMPLE IX Multiple Sclerosis Vaccine

A. Human MBP-reactive T cells

MBP-reactive T cell lines were established from peripheral bloodmononuclear cells (PBMC) of nine chronic progressive MS patients and twohealthy controls. Cells were maintained in culture by regularstimulation with purified human MBP and irradiated-autologous PBMC forthree days followed by four days in IL-2 containing medium.

B. PCR Amplification of TCR β-chain genes from MBP-reactive T cell lines

T cells were harvested from log phase cultures and RNA was prepared,amplified with the Vβ16mer primer and nested Cβ primers for 55 cycles asdescribed in Example X.

C. TCR β-chain sequences of human MBP-reactive T cells

Vβ16mer amplified TCR β-chain genes from human MBP-reactive T cell lineswere sequenced using the Cβseq primer. Amplification products were gelpurified, base denatured and sequenced from the Cβseq primer. ReadableDNA sequence was obtained from 5 of these lines, indicating thatpredominant T cell clones had been selected by long term in vitropassage. One of these sequences, from the MS-Re cell line (Table 11),possessed a β-chain VDJ amino acid sequence that shared five of thefirst six and six of nine total residues with the β-chain VDJ amino acidsequence conserved among MBP reactive, encephalitogenic T cells in theB10.PL mouse model of EAE. This sequence was not present among thepredominant TCR rearrangements found in the remaining four human MBPreactive T cell lines.

To determine if similar sequences were present in the β-chain repertoireof the MBP-reactive T cell lines from other MS patients, PCRamplification was conducted with a degenerate (n=1024) 21-nucleotideprimer (VβRe) corresponding to seven amino acids of this sequence. RNAswere reversed transcribed and amplified in 20 cycle stage I reactionswith the Vβ16mer and Cβext primers. One μl aliquots of these stage Ireactions were reamplified for 35 cycles with the VβRe and Cβ intprimers. One μl aliquots of these reactions were analyzed by Southernblot hybridization with a ³²P-labeled human Cβ probe. This analysisrevealed the 300 bp amplified product in the Re cell line and in one ofthe other MS patient lines, but not in MBP-reactive T cells from controlsubjects or in non-MBP reactive human T cell lines and clones. Thepresence of this sequence in two of the nine MS patient lines tested iscompelling. Since this sequence is known to be conserved amongencephalitogenic T cells in EAE, its detection among MBP-reactive Tcells from MS patients demonstrates a role for T cells bearing thisdeterminant in the pathogenesis of MS.

Immunogenic peptides having the sequence SGDQGGNE (SEQ ID No.1) can besynthesized as shown in Example II and used to immunize human subjectsby methods demonstrated in Example III. Such immunizations can result inan effective immune response.

TABLE 11 Seq ID No. A) Sample Vβ Dβ Jβ     Vβ4.2                            Jβ2.1 MS-Rectctgc  agcggagaccagggcggc  aatgagcagttcttc 32         S  G  D  Q  G  G -  N  E  Q  F  F 33 B10.PL         S  G  D  A  G  G  G Y  E 23 B)        A                 A     A34        C     C     A     C     C     C     A 355′ G G   G A   C A   G G   G G   A A   G A   3′       G     T     G     G     G     T     G 36       T                 T     T 37

EXAMPLE X Detection of Clonal Infiltrates of Activated Vβ17 T Cells inthe Synovium of Rheumatoid Arthritis Patients

A. T cell preparations from synovial tissue

Synovial tissue specimens were obtained from radiographically provenrheumatoid arthritis patients undergoing joint replacement therapy.Activated T cells were selected using magnetic beads and antibodiesreactive with the human IL2-R (αIL2-R) as follows. Synovial tissue wasdigested for 4 hrs at 37° C. in RPMI +10% Fetal Bovine Serum (FBS)containing 4 mg/ml collagenase (Worthington Biochemical, Freehold, N.J.)and 0.15 mg/ml DNAse (Sigma, St. Louis, Mo.). Digests were passedthrough an 80-mesh screen and single cells were collected by Ficolldensity gradient centrifugation. Cells at the interface were washed andwere incubated at 10⁶/ml for 30 min at 0° C. with 5 μg/ml control mouseIgG (Coulter Immunology, Hialeah, Fla.) in PBS containing 2% FBS(PBS-FBS). Cells were washed three times and incubated for 30 min at 0°C. with magnetic beads conjugated to goat anti-mouse IgG (AdvancedMagnetics, Cambridge, Mass.). Beads were magnetically separated andwashed three times with PBS-FBS. This preselection with mouse IgG (mIgG)and magnetic beads was used to control for non-specific adsorption of Tcells. The cells remaining in the initial suspension were furtherincubated 30 minutes at 0° C. with 5 μg/ml monoclonal mouse IgG reactivewith the human T cell IL2-R (Coulter Immunology, Hialeah, Fla.). Cellswere washed and selected with magnetic beads as above. Beads from theIgG preadsorption and the IL2-R antibody selection were immediatelyresuspended in acidified-guanidinium-phenol-chloroform and RNA preparedas described in Chonezynski and Sacchi, Anal. Biochem. 162:156 (1987),which is incorporated herein by reference. Since RNAS were preparedwithout in vitro culture of the cells and the accompanying bias that maybe induced, they are expected to accurately reflect T cell distributionsin synovial tissue at the time of surgical removal. Only half of themIgG and αIL2-R beads from patient 1012 were immediately processed forRNA. The remainder were cultured for 5 days in RPMI 1640, 5% FBS, 20%HL-1 (Ventrex Laboratories Inc., Portland, Me.), 25 mM HEPES, glutamine,antibiotics and 20% LAK supernatant (Allegretta et al., Science, 247:718(1990)), which is incorporated by reference herein, as a source of IL-2.RNA was extracted from cultures of the αIL2-R beads (1012IL2.d5), butnot from the 1012mIgG sample as no viable cells were present at the endof the 5 day culture.

A T cell clone was derived from the Ficoll pellet of patient 1008. Thecells in the pellet were cultured at 2×10⁶/ml in media without IL-2 fortwo weeks. Non-adherent cells from this culture were cloned by limitingdilution onto autologous synovial cell monolayers. A CD4+ T cell clone1008.8 was obtained and adapted to culture by regular stimulation withautologous synovial monolayers for 3 days in media without IL-2 followedby a 4 day culture in medium with LAK supernatant.

B. Lysis of Synovial Adherent Cells by 1008.8

Lysis of synovial adherent cells by 1008.8 was demonstrated as follows.Synovial cell monolayers were labeled as described in Stedman andCampbell, J. Immunol. Meth. 119:291 (1989), which is incorporated hereinby reference, with ³⁵S for use as targets in CTL assays. Cells weretypsinized, washed and plated at 2000 cells per well of a 96-well roundbottom microtiter plate. 1008.8 cells, cultured for 3 days prior to theassay with synovial adherent cells and medium containing LAKsupernatant, were added to the targets at the indicated effector:targetratios. Cultures were incubated overnight at 37° C., centrifuged at 300×g for 2 minutes and radioactivity in 50 μl of the supernatantquantified. Per cent specific lysis was calculated relative todetergent-lysed targets by standard formulas. This clone is cytotoxicfor synovial adherent cell targets in CTL assays (Table 12).

TABLE 12 Effector:Target Ratio % Specific Lysis  5:1  7 10:1 16 25:1 32

C. PCR Amplification of TCR β-chain genes

TCR β-chain genes were amplified with several combinations of theprimers shown in FIG. 2. The vβ16mer primer is a degenerate Vβ primer(n=256) which is predicted to bind 85% of human TCR β-chain genes at all16 residues and 95% at 15 residues. This primer has been used to amplifyTCR β-chains from more than 25 different human T cell clones, lines orprimary tissue preparations. A spectrum of Vβ genes has been sequencedfrom these amplified DNAs, arguing against a significant bias of theprimer for certain Vβ families. Thus, PCR amplification with the Vβ16merprimer facilitates analysis of T cell populations for which a prioriknowledge of Vβ gene usage is unavailable.

T cell receptor β-chain genes were amplified in two-stage amplificationreactions with nested pairs of the primers shown in FIG. 2. The primersequences used in the polymerase chain reactions are listed in Table 13.

TABLE 13     G AC     CAAA (SEQ ID Vβcons 5′ T TC TGGTA    CA 3′ NO. 47)    T TT     TCGT (SEQ ID NO. 48) Vβ17 5′ TCACAGATAGTAAATGACTTTCAG 3′(SEQ ID NO. 49) Vβ8 5′ TCTCCACTCTGAAGATCC 3′ (SEQ ID NO. 50) Vβ125′ GATTTCCTCCTCACTCTG 3′ (SEQ ID NO. 51) 5′Cβ 5′ CAAGCTGTTCCCACCCGA 3′(SEQ ID NO. 52) Cβext 5′ CCAGAAGGTGGCCGAGAC 3′ (SEQ ID NO. 53) Cβint5′ GCGGCTGCTCAGGCAGTA 3′ (SEQ ID NO. 54) Cβseq 5′ CGACCTCGGGTGGGAACA 3′(SEQ ID NO. 55)

RNAs were reverse transcribed for 1 hour at 42° C. with 40 pmol of theCβext primer in a 12 μl reaction using conditions described by Hart etal., The Lancet, p. 596 (1988), included by reference herein. Reactionswere diluted with a master mix containing 40 pmols of the Vβ16merprimer, nucleotides and reaction buffer as above but without MgCl₂ togive a final Mg⁺² concentration of 3.6 mM. Samples were denatured for 15minutes at 95° C., 1 unit of heat stable recombinant DNA polymerase(Cetus Corporation, Emeryville, Calif., Ampli-taq™) was added and 20cycles of PCR conducted. Each cycle consisted of a 1 min denaturation at95° C., a two minute annealing step and a two minute extension at 72° C.The first two cycles were annealed at 37° C. and 45° C., respectively,and the remainder at 50° C. One microliter aliquots of these stage Ireactions were added to 100 μl stage II amplification reactions (Cetus,Gene-Amp Kit™) containing 100 pmols of the Cβint primer and 100 pmols ofthe Vβ8, Vβ17 or 5′Cβ primers or 700 pmols of the Vβ16mer primer. StageII amplifications were conducted as above with a 50° C. annealingtemperature and without the 37° C. and 45° C. ramping.

RNA samples from 1012IL2.d5 and 1008.8 cultures were amplified with theVβ16mer and Cβext primers in stage I reactions and with the Vβ16mer andthe Cβint primer in 35 cycle stage II reactions. Reaction products,purified from low melting agarose gel slices with Gene Clean glass beads(Bio 1D1, San Diego, Calif.), were base denatured and sequenced from theCβseq primer with T7 polymerase (Sequenase, United States Biochem,Cleveland, Ohio). A predominate Vβ sequence, corresponding to a singleVβ17 rearrangement (Table 14), was clearly readable in the 1012IL2.d5sample. Other, less frequent rearrangements were detected as faint,uninterpretable background bands in the sequencing gels. Culture ofthese 1012.IL2 cells in IL2-containing medium without added accessorycells or antigen is not expected to induce de novo activation of these Tcells. Thus, the predominance of a single Vβ17 rearrangement in thissample reflects in vivo clonal expansion of Vβ17+ T cells in thispatient. DNA sequence determination of TCR β-chain DNA amplified fromthe cytotoxic T cell clone, 1008.8, also revealed a Vβ17 rearrangement(Table 14). The presence of Vβ17 rearrangements in these two differenttypes of synovial T cell samples, derived from two separate RA patients,implicates Vβ17 bearing T cells in the pathogenesis of RA.

TABLE 14 SEQ ID Sample Vβ Dβ Jβ NO. 1012 Y L C A S K N P T V S Y G Y T F38 day 5 tatctctgtgccagt aaaaatcccacggtctcc tatggctacaccttc 39 Vβ17Jβ1.2 Y L C A S D N E S F F G Q G 40 1008.8 tatctctgtgccagt gacaacgagagtttctttggacaaggc 41 Vβ17 Jβ1.1 1014 Y L C A S V R D R R N Y G Y T 42 IL-2tatctctgtgccagt gtgagggacaggaga aactatggctacacc 43 Vβ17 Jβ1.2 1015 Y L CA S S S I D S S Y E Q Y 44 IL-2 tatctctgtgccagtagt agtatagactcctcctacgagcagtac 45 Vβ17 Jβ2.7

To determine whether or not Vβ17 rearrangements were present in theother magnetic bead RNA preparations, TCR β-chain genes were amplifiedwith a Vβ17-specific primer in the second stage amplification after aninitial amplification with the Vβ16mer. Vβ17 TCR DNA could be amplifiedfrom magnetic bead samples derived from the 4 patients examined.Ethidium bromide staining of electrophoresed reaction products revealedgreater Vβ17 amplification in some of the IL-2R+ samples than in thecorresponding controls. Accordingly, the relative amounts of Vβ17 TCRsin each control and IL-2R+ sample were quantified by slot blothybridization analysis as follows.

RNAs from magnetic bead preps were amplified in the first stage with theVβ16mer and Cβext primers and then reamplified for twenty cycles withthe Cβint primer and each of the Vβ17, Bβ8 and 5′Cβ primers.Amplification reactions were serially diluted in 20× SSC, denatured byboiling and chilled in an ice slurry. Samples were loaded ontonitrocellulose membranes, hybridized to a human TCR β-chain constantregion probe and washed with 0.1× SSC, 0.1% SDS at 56° C. Boundradioactivity was quantified by liquid scintillation spectroscopy andendpoint dilutions were those samples with fewer than 200 cpm bound. Theamounts of product produced by forty total cycles with each of therespective primer combinations falls in the linear portion of a productversus cycle number quantification curve.

Amplifications with the 5′Cβ and Cβint primer pair were used to estimatethe total β-chain amplified from each sample, providing benchmarks fornormalizing the results of Vβ17 and Vβ8 quantification in the respectiveIL-2R+ and control sample pairs (Table 15). The quantity of Vβ17 DNAamplified was increased in the IL-2R+ samples, relative to the controlsamples, in 3 of the 4 patients. The magnitude of the increase rangedfrom 5-fold in patient 1015 to 40-fold in patient 1014 (Table 15). Thisenrichment was not a product of the isolation procedure, since thequantity of Vβ8 DNA amplified was increased in the IL-2R+ fraction onlyin patient 1015.

TABLE 15 Endpoint Dilution Vβ17IL-2R Vβ8IL-2 Sample Cβ Vβ17 Vβ8 Vβ17/CβmIgG Vβ8/Cβ mIgG 1 3,125 3,125 625 1 25 0.2 1   2 3,125 125 625 0.04 0.23 15,625 25 625 0.001 0.12 0.04 0.04 4 3,125 25 3,125 0.008 1 5 15,625625 125 0.04 40 0.008 0.04 6 3,125 5 625 0.001 0.2 7 15,625 625 15,6250.04 5 1 25*   8 78,125 625 3,125 0.008 0.04 Sample 1 = 1012 IL-2R+,Sample 2 = 1012 mIgG, Sample 3 = 1013 IL-2R+, Sample 4 = 1013 mIgG,Sample 5 = 1014 IL-2R+, Sample 6 = 1014 mIgG, Sample 7 = 1015 IL-2R+,Sample 8 = 1015 mIgG. *In a follow-up study in which Vβ8 was sequenced,it was found that the enrichment of this Vβ was an artifact and that thevalue is ≦1.

Vβ17 rearrangements from the IL-2R+ RNAs of the three patients showingenrichment were amplified with the Vβ17 and Cβint primer pair and thereaction products sequenced with the Cβseq primer. As was shown forsample 1012 IL-2.d5, 1014 and 1015 contained single sequences (Table14), indicative of clonal expansion of Vβ17 T cells in vivo. Incontrast, direct sequencing of the rearrangements amplified with the Vβ8specific primer was not possible due to significant heterogeneity in theβ-chain product.

D. HLA-DR Analysis in Rheumatoid Arthritis Patients

HLA-DR analysis in rheumatoid arthritis patients was performed asfollows. DNA from each patient was prepared by boiling 10⁵ synovialcells in 200 μl dH₂O. Ten μl were amplified for 35 cycles in a 100 μlreaction (Cetus, GENE AMP KIT™) containing 100 pmols of each of the DRβPCR primers shown in Table 16 as DRβ1 (SEQ ID NO:56) and (SEQ ID NO:57)and DRβ3 (SEQ ID NO:69), (SEQ ID NO:70) and (SEQ ID NO:71). One-tenth μlof this reaction was reamplified in 10 μls containing only the DRβ2 (SEQID NO:58) and (SEQ ID NO:59) primer and 17 pmol of α32P-dCTP as the solesource of dCTP for 10 cycles. Reactions were spiked with 200 μM dCTP andchased for 2 cycles. The resulting negative strand probes werehybridized to slot blots containing 10 pmol of the HLA-DR allelespecific oligos (positive strands) using conditions previously describedby Amar et al., J. Immunol. 138:1947 (1987), which is incorporatedherein by reference. The slots were washed twice for 20 minutes withtetramethylammoniumchloride (Wood et al., Proc. Natl. Acad. Sci. USA82:1585 (1985)) which is incorporated herein by reference) at 65-68° C.and exposed to X-ray film.

Each of the patients in this study possessed at least one allele of theHLA-DR genes, DR4w4, DR1, DR4w14 or DR4w15, that are known to predisposefor RA (Table 16). Also shown in Table 16 are HLA-DR allele specificoligonucleotides.

TABLE 16 SEQ ID NO.       A 56 DRβ1 5′ G A G T   C T G G A A C A G C 3′      C 57             A 58 DRβ2 5′ G T A G T T G   T T C T G C A 3′            G 59 HLA-DR ALLELE-SPECIFIC OLIGONUCLEOTIDES DRβ1 GenesDR1,DR4w14,DR4w15 5′ CTC CTC GAG CAG AGG CGG GCC GCG 3′ 60 DR2*5′ T-- --- --- G-C --- --C --- --- 3′ 61 DR35′ --- --- --- --- -A- --- -G- CG- 3′ 62 DR4w45′ --- --- --- --- -A- --- --- --- 3′ 63 DR4w135′ --- --- --- --- --- --- --- -A- 3′ 64 DR5, DR6, DR4w105′ A-- --- --A G-C GA- --- --- --- 3′ 65 DR75′ A-- --- --- G-C --- --- -G- CA- 3′ 66 DR85′ T-- --- --A G-C --- --- --- CT- 3′ 67 DR95′ T-- --- --- -G- --- --- --- -A- 3′ 68 DRB3 Genes DR25′ A-- --- --- --- GC- --- --- --- 3′ 69 DR35′ --- --- --- --- -A- --- -G- CAG 3′ 70 DR7, DR95′ --- --- --- -G- --- --- --- -A- 3′ 71 Patient HLA-DR 1008 1,4w4 10121,3 1013 1,7 1014 1,4w4 1015 4w4,4w4 *The dashes refer to amino acids incommon with DR1, DR4w14, DR4w15

T cell receptors containing Vβ17 or fragments thereof which areimmunogenic or can be made immunogenic can be used to immunize humansubjects by methods demonstrated by Example VIII. Such immunizations canresult in an effective immune response.

EXAMPLE XI

Synovial tissue specimens were obtained from proven RA patientsundergoing joint replacement surgery. HLA-DR analysis was conducted asdescribed in Example IX(D).

A. Polymerase chain reaction (PCR) amplification of T cell receptorβ-chain genes

T cell receptor β-chain genes were amplified in two-stage amplificationreactions with nested pairs of HPLC-purified oligonucleotide primers(Midland Certified Reagents, Midland, Tex.) shown in FIG. 3. RNAs werereverse transcribed (1 hour, 42° C.) with the Cβext primer (40 pmol) in12 μl of reaction buffer (Hart et al., Lancet ii:596-599 (1988)).Reactions were diluted with a master mix (8 μl) containing the Vβconsprimer (40 pmol), nucleotides and reaction buffer minus MgCl₂ (final Mgconcentration=3.6 mM). Samples were denatured (15 minutes, 95° C.) and20 cycles of PCR were conducted using Taq polymerase (1 unit, CetusAMPLI-TAQ™). Each cycle consisted of a 1 minute denaturation at 95° C.,a two minute annealing step and a two minute extension at 72° C. Thefirst two cycles were annealed at 37° C. and 45° C. and the remainder at50° C. One microliter aliquots of these Stage I reactions were added to100 μl Stage II amplification reactions (Cetus Gene-Amp Kit) containingthe Cβint primer (100 pmol) and either the Vβ8, Vβ12, Vβ17, or 5′Cβprimers (100 pmol) or the Vβcons primer (100-700 pmol). Stage IIamplifications were conducted for the indicated number of cycles with a50° C. annealing temperature and without the 37° C. and 45° C. ramping.

B. Vβ17 T cells are cytotoxic for synovial adherent cells

Two parallel cultures were established from single cell suspensions,derived by enzymatic digestion as described below from the synovialtissue of patient 1008. The first, a bulk culture of total synovialtissue cells, was plated at 2×10⁶/ml in RPMI 1640, 5% FBS, 20% HL-1(Ventrex Laboratories, Portland, Me.), 25 mM HEPES, glutamine andantibiotics. The culture was grown undisturbed for two weeks in theabsence of exogenous antigen or growth factors. The second, a synovialcell monolayer culture, was initiated as above and regularly trypsinizedand passaged during this two-week period. Monolayer cells were plated at5×10⁴ cells/well in flat-bottomed, 96-well microtiter plates andcultured overnight. Non-adherent cells from the total synovial cellculture were then plated at 10 cells/well onto the monolayers. Wellspositive for T cell growth were expanded and adapted to culture byregular stimulation with autologous synovial monolayers for 3 days inmedia without IL-2 followed by a 4 day culture in medium containing 20%supernatant from lymphokine activated killer (LAK) cells, a source ofIL-2 (Allegretta et al., Science 247:718-721 (1990)). Later passageswere adapted to weekly stimulation with allogeneic PBLs and anti-CD3antibody (Coulter Immunology, Hialeah, Fla.) in place of synovial cellmonolayers.

Cytotoxicity assays were conducted as described Stedman et al., Immunol.Meth. 119:291-294 (1989), incorporated herein by reference, using³⁵S-labeled synovial monolayer cells or EBV-transformed B cells astargets. Target cells were labeled overnight, trypsinized (adherentcells only), washed and plated at 2000 cells per well in 96-well roundbottom microtiter plates. T cells were activated with allogenic PBLs andanti-CD3 antibody in medium containing LAK supernatant for 7 days priorto the assay and added to the targets at the indicated effector:targetratios. Cultures were incubated overnight at 37° C., centrifuged at 300×g for 2 minutes and radioactivity in 50 μl of the supernatantquantified.

The per cent specific lysis was calculated relative to detergent-lysedtargets as described in Townsend et al., Cell 44:959-968 (1986),incorporated herein by reference. At effector:target ratios of 1.0, 2.5and 5.0, the percent lysis of synovial adherent cells by 1008.8 T cellswas approximately 4%, 14% and 33%, respectively. By comparison, at thesame effector:target ratios, 1008.8 T cells had no demonstrated effecton EBV-transformed B cell targets. Similarly, at the sameeffector:target ratios, the percent lysis of synovial adherent cells byMS3 cells was approximately 0%, 0% and 3%, respectively.

T cells were isolated from the synovial tissue of patient 1008 byco-cultivation with synovial cell monolayers. Since the antigensrecognized by pathogenic T cells in RA are unknown, synovial cellmonolayers were used as stimulators of in vitro synovial T cell growth.The relevant target cells were assumed to be present in a bulk adherentcell culture from diseased synovium. Of 192 microwell co-culturesplated, 7 were positive for T cell growth within 10-14 days. T cellswere expanded and maintained in vitro by alternately stimulating withsynovial cell monolayers and medium containing LAK supernatant. Flowcytometry revealed that each of these seven cultures was 100% CD4+. Oneculture designated 1008.8, grew especially vigorously and, as assessedmicroscopically promoted the destruction of the monolayer cells. CTLassays confirmed the cytotoxicity of 1008.8 for these monolayer targetsas discussed above. Cytolysis was specific for synovial cell targets, asno lysis of autologous Epstein-Barr virus-transformed B cells wasdemonstrable. Neither of the targets was lysed by a CD4+, myelin basicprotein-reactive human CTL clone, MS3, excluding the possibility thatthe monolayer cells were susceptible to lysis by any activated T cell.In repeated assays with 1008.8, specific lysis never exceeded 30-35%,suggesting that the relevant synovial target cell comprisesapproximately that proportion of the total monolayer culture, whichbased on morphology is a mixture of multiple cell types.

The T cell receptor (TCR) β-chain gene of 1008.8 was amplified by thepolymerase chain reaction (PCR) as described in Mullis et al., Meth.Enzym. 155:335-350 (1987), incorporated herein by reference, and its DNAsequence was determined. Amplification was accomplished using aconsensus Vβ primer (Vβcons), a degenerate 16 nucleotide primer (n=256)which is homologous at all 16 residues with 78%, and at 15 of 16residues with 98%, of known human TCR Vβ genes, which have been compiledby Kimura et al., J. Immol. 17:375-383 (1987). This primer was designedto amplify β-chain rearrangements containing any of the known Vβ genes,thus allowing analysis of T cell clones for which a priori knowledge ofVβ gene usage is unavailable. Sequencing of the Vβcons-amplified β-chaingene of 1008.8 revealed a single Vβ17-Jβ1.1 rearrangement as shown inTable 16. Subsequently, the remaining six cultures derived from patient1008 were analyzed by PCR amplification using a Vβ17-specific primershown in Table 13. TCR genes from three of those six were amplifiable,indicating that, of the 7 T cell cultures obtained from the synovium ofthis patient, 4 had rearranged and expressed the Vβ17 gene.

C. Vβ17 T cells are enriched among activated T cells in RA synovium

Synovial tissue was digested with agitation for 4 hrs at 37° C. inRPMI-1640 and 10% fetal bovine serum (FBS) containing 4 mg/mlcollagenase (Worthington Biochemicals, Freehold, N.J.) and 0.15 mg/mlDNAse (Sigma Chemical, St. Louis, Mo.). Digests were passed through an80-mesh screen and single cells were collected from the interface ofFicoll density gradients, washed and incubated at 10⁶/ml for 30 min at0° C. with 5 μg/ml control mouse IgG (Coulter) in PBS containing 2% BSA.Cells were washed 3× and incubated for 30 min at 0° C. with magneticbeads conjugated to goat anti-mouse IgG (Advanced Magnetics, Cambridge,Mass.). After magnetic removal of the beads, the remaining cells wereincubated 30 minutes at 0° C. with 5 μg/ml mouse anti-human IL-2R(Coulter), washed and selected with magnetic beads as above. Cell-coatedbeads from the mIgG preadsorption and the IL-2R antibody selection werewashed 3×, immediately resuspended inacidified-guanidinium-phenol-chloroform and RNA was prepared asdescribed in Chomczynski et al., Anal. Biochem. 162:156-159 (1987).

RNAs from magnetic bead preparations were reverse transcribed andamplified for 20 cycles in stage I reactions with the Vβcons and Cβextprimers. One μl of each reaction was reamplified for 20 cycles inindividual stage II reactions containing the Cβint primer in conjunctionwith the Vβ17, Vβ8, Vβ12 and 5′Cβ primers. Aliquots of each reactionwere diluted in 20× SSC, denatured by boiling and chilled in an iceslurry. Samples were loaded onto nitrocellulose membranes, hybridized toa human TCR β-chain constant region probe and washed with 0.1× SSC, 0.1%SDS at 56° C. Bound radioactivity was quantified by liquid scintillationspectroscopy. The amounts of product produced by 40 total cycles witheach of the respective primer combinations falls in the linear portionof a product versus cycle number quantification curve. Values shown inTable 17 below reflect the relative increase or decrease for thespecific Vβs in the IL2-R+ versus mIgG controls calculated according tothe formula:$\frac{{specific}\quad V\quad \beta \quad {cpms}\quad {\left( {{IL2} - R +} \right)/C}\quad \beta \quad {cpms}\quad \left( {{IL2} - R +} \right)}{{specific}\quad V\quad \beta \quad {cpms}\quad {({mIgG})/C}\quad \beta \quad {cpms}\quad {({mIgG}).}}$

TABLE 17 Ratio IL−2R+ Sample     mIgG  Experiment # Vβ17 Vβ8 Vβ12 1012 11.88 0.42 1.35 2 1.60 0.39 0.72 3 2.11 0.48 0.64 X^(˜) ± S.D. 1.86 ±0.25 0.43 ± 0.04 0.900 ± 0.38 1013 1 2.54 0.49 N.D. 2 3.65 0.87 0.87 34.29 2.07 0.96 X^(˜) ± S.D. 3.49 ± 0.88 1.14 ± 0.82 0.91 ± 0.06 1014 14.70 0.17 N.D. 2 1.68 0.10 1.50 3 1.92 0.09 0.44 4 2.34 0.07 0.55 X^(˜)± S.D. 2.66 ± l.38 0.10 ± 0.04 0.83 ± 0.58 1015 1 3.40 0.20 0.85 2 2.850.47 1.82 X^(˜) ± S.D. 3.12 ± 0.50 0.38 ± 0.15 1.33 ± 0.68

Next, the presence of Vβ17 T cells in the synovial tissue of other RApatients was determined. Since the rheumatoid synovium contains amixture of activated and non-activated T cells, the activated T cellswere identified as the most relevant for the initiation and perpetuationof the disease pathogenesis. Thus, activated T cells from single-cellsuspensions of synovial tissue were selected using magnetic beads andantibodies reactive with the human interleukin-2 receptor (IL-2R). Cellsuspensions from each patient were pretreated with an isotype-matchedmouse IgG (mIgG) and magnetic beads to control for non-specificadsorption. RNAs were directly extracted from cells in the IL-2R+ andcontrol samples without in vitro culture and, therefore, are expected toaccurately reflect T cell distributions in synovial tissue at the timeof surgical removal.

The initial PCR amplifications of these magnetic bead RNA preparationsrevealed greater amounts of Vβ17 PCR product in the IL2-R+ samples thanin the corresponding mIgG controls. The presence of TCR mRNA in the mIgGsamples indicates that T cells non-specifically adhered to the magneticbeads. The apparent increase Vβ17 PCR product in the IL2-R+ samplesuggests that the activated T cell compartment contained more Vβ17 Tcells than the unselected synovial T cell compartment. Thus, aquantitative PCR analysis was used to formally examine the relativeproportions of Vβ17 T cells in the IL-2R+ and mIgG control samples(Table 17). Magnetic bead RNAs were reverse transcribed, preamplifiedwith Vβcon and Cβext and reamplified in separate reactions with aconstant region primer (Cβint) and each of the Vβ-specific primers,Vβ17, Vβ8 and Vβ12. The second stage amplification was also performedwith two Cβ primers (5′Cβ and Cβint) in order to estimate the totalβ-chain present in each sample and to provide benchmarks for normalizingthe results of specific Vβ quantification in the respective IL-2R+ andcontrol sample pairs. The proportion of Vβ17 DNA, relative to total Cβ,was increased in the IL-2R+ samples over that found in the mIgG controlsamples for each of the 5 patients examined. This increase was observedin multiple analyses and the means are shown in Table 17. Enrichment wasnot a product of the isolation procedure, since the quantity of Vβ8 orVβ12 DNA amplified was not significantly increased in the IL-2R+fraction of any of the patients. Thus, activated T cells in therheumatoid synovium do not represent a cross section of all possible Vβfamilies, but preferentially contain Vβ17 T cells, and possibly other Vβfamilies which were not quantified in this analysis.

D. Synovial Vβ17 T cells display limited heterogeneity RNAs were reversetranscribed and amplified

with the Vβcons and Cβext primers in 20 cycle stage I reactions and withthe Cβint and Vβcons (1008.8) or Vβ8, Vβ12, and Vβ17 primers (magneticbead RNA preparations) in 35 cycle stage II reactions. Double strandedreaction products were electrophoresed in 2% Nu-Sieve agarose gels.After purification from gel slices with Gene Clean (Bio 101, San Diego,Calif.), samples were base denatured and either directly sequenced orcloned into plasmid for sequencing of multiple independentrearrangements. In all cases, samples were sequenced from the Cβseqprimer (FIG. 2) with T7 polymerase (Sequenase, United StatesBiochemicals, Cleveland, Ohio).

Vβ17 rearrangements present in the IL-2R+ RNAs were amplified from theVβcons and Cβext preamplification with the Vβ17 and Cβint primer pairand the reaction products sequenced. PCR products from patients 1014 and1015 were directly sequenced and the results obtained were consistentwith the presence of a single Vβ17 rearrangement in each of theseamplified samples (Table 17). The PCR product of patient 1013 was clonedinto plasmid and the thirteen isolates that were sequenced containedidentical Vβ17 rearrangements. Sequencing of plasmid clones from patient1012 revealed the presence of two dominant rearrangements (5 isolates ofeach) and a single isolate of another. Thus, the Vβ17 repertoire in theRA synovium is of limited heterogeneity, indicative of clonal oroligoclonal expansion of Vβ17 T cells in vivo. This is in contrast tothe Vβ8 and Vβ12 repertoires, from these same synovial preparations,which showed significant heterogeneity. None of the PCR-amplified Vβ8and Vβ12 samples analyzed were directly sequenceable and plasmid cloningof Vβ8 rearrangements from patient 1012 revealed 4 different sequencesin 5 clones analyzed.

Vβ17 rearrangements in PBLs from patient 1012 also revealed greaterdiversity than that seen in the synovial IL-2R+ sample. RNA from a 3 dayculture of PHA/PMA stimulated 1012 PBLs was amplified with the Vβ17primer, as for the synovial sample, and the products cloned intoplasmid. Nine different rearrangements, none of which corresponded tothose present in the 1012 synovium, were found in the 10 clones thatwere sequenced. Thus, the restricted heterogeneity of Vβ17rearrangements in the activated synovial T cell population of patient1012, as well as the other patients examined, likely results, not fromrandom T cell trafficking, but from the selective expansion of thoseVβ17-bearing T cells in the diseased tissue.

EXAMPLE XII

A. Detection of TCR β-chain transcripts in synovial T cells usingVβ-specific PCR-amplification

T cell receptor β-chain genes were amplified in two-stage reactions withindividual Vβ-specific primers as described in Wucherfpennig et al.,Science, 248:1016-1019 (1990), incorporated herein by reference, andnested Cβ primers. RNAs were reverse transcribed (1 hour, 42° C.) withthe Cβext primer (40 pmol) in 12 μl of reaction buffer (Hart et al.,supra). Reactions were diluted with a master mix (8 μl) containingnucleotides, reaction buffer minus MgCl₂ (final Mg⁺² concentration=3.6mM) and Taq polymerase, apportioned among 19 tubes containing theindividual Vβ primers. Samples were denatured (15 minutes, 95° C.) and20 cycles of PCR were conducted. Each cycle consisted of a one minutedenaturation at 95° C., a two minute annealing step and a two minuteextension at 72° C. The first two cycles were annealed at 37° C. and 45°C. and the remainder at 50° C. One microliter aliquots of these Stage Ireactions were added to 100 μl Stage II amplification reactions (CetusGene-Amp Kit) containing the Cβint primer (100 pmol) and 100 pmol of theVβ primers used in the corresponding preamplifications. Stage IIamplifications were conducted for 20 cycles with a 50° C. annealingtemperature and without the 37° C. and 45° C. ramping. Five μl of eachreaction was electrophoresed in 2% agarose gels, transferred tonitrocellulose and hybridized to a ³²P-labeled β-chain constant regionprobe. Blots were exposed to X-ray film and scored for the presence orabsence of Vβ-specific amplification.

B. Vβ17, Vβ14, Vβ9 and Vβ3 transcripts are common among activatedsynovial T cells

To ensure that the prevalence of Vβ17 rearrangements in the earlierstudies did not result from an amplification bias of the Vβ16mer primer,and to assess the presence of other Vβ genes in the synovium, TCRtranscripts in the IL2-R+ samples were analyzed with a panel of 19 PCRprimers, specific for known human Vβ gene families (Table 18). TheVβcons primer of Example XI is equivalent to the Vβ16mer primer. Thenumber of Vβ genes detectable in these samples was variable, rangingfrom two to twelve. Vβ17 was found in four of the five patients,confirming the previous analyses using the Vβ16mer primer. Vβ14transcripts also were found in four of the five patients and Vβ3 and Vβ9transcripts were detectable in three of the five samples. Thus, T cellsbearing these 3 Vβ polypeptides may also contribute to synovialinflammation.

TABLE 18 Analysis of IL-2R+ Synovial T Cells with Individual Vβ-specificPrimers Vβ Families Patient # 1 2 3 4 5 6 7 8 9 10 11 12 14 15 16 17 1819 20 1012 + + + 1013 + + + 1014 + + 1015 + + + + + + + + + + + +1020 + + + + + + + + + +

EXAMPLE XIII Vaccination Against EAE with a CDR4 Peptide of Vβ8.2

Rats were immunized with 100 μg of a synthetic peptide containing thesequence VPNGYKVSRPSQGDFFLTL (SEQ ID No.46) found in the fourthhypervariable or CDR4 region of the rat TCR β chain identified as Vβ8.2.The peptide, dissolved in saline, was emulsified in an equal volume ofcomplete Freund's adjuvant (CFA) containing 10 mg/ul of mycobacterialtuberculosis. The animals were challenged 30 days later with 50 μg ofguinea pig myelin basic protein in CFA. The CDR4 peptide vaccinationresulted in a reduced incidence of disease as well as a reduced severityas measured both clinically and histologically as shown in Table 19. Thehistology was performed essentially as described in Hughes & Powell, J.Neuropath. Exp. Neurol. 43:154 (1984), which is incorporated herein byreference.

TABLE 19 Protection from EAE in the Rat by Vaccination with a TCR-CDR4Peptide¹ Mean #Clinical Signs Mean Max Mean Mean #With HistologyHistology Vaccination Challenge #Tested Severity² Onset Duration #TestedScore³ Rat CDR4 50 μg 6/10 1.5 11.8 5.4  3/10 0.6 in CFA MBP/CFA PBS/CFA50 μg 9/10 2.5 12.1 6.1 6/9 1.4 MBP/CFA None 50 μg 10/10  3.0 10.5 7.39/9 3.0 MBP/CFA ¹rat CDR4 peptide sequence: VPNGYKVSRPSQGDFFLTL (SEQ IDNo.46) ²graded on a 3-point scale as described in Example I ³graded on a4-point scale

Although the invention has been described with reference to thepresently-preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

75 8 amino acids amino acid linear peptide N-terminal unknown 1 Ser GlyAsp Gln Gly Gly Asn Glu 1 5 8 amino acids amino acid linear peptideN-terminal unknown 2 Cys Ala Ile Gly Ser Asn Thr Glu 1 5 11 amino acidsamino acid linear peptide N-terminal unknown 3 Ala Ser Ser Leu Gly GlyAla Val Ser Tyr Asn 1 5 10 12 amino acids amino acid linear peptideN-terminal unknown 4 Ala Ser Ser Leu Gly Gly Glu Glu Thr Gln Tyr Phe 1 510 12 amino acids amino acid linear peptide N-terminal unknown 5 Ala SerSer Leu Gly Gly Phe Glu Thr Gln Tyr Phe 1 5 10 11 amino acids amino acidlinear peptide N-terminal unknown 6 Ala Ser Ser Leu Gly Gly Thr Glu AlaPhe Phe 1 5 10 113 amino acids amino acid linear peptide N-terminalunknown 7 Met Ser Asn Gln Val Leu Cys Cys Val Val Leu Cys Phe Leu GlyAla 1 5 10 15 Asn Thr Val Asp Gly Gly Ile Thr Gln Ser Pro Lys Tyr LeuPhe Arg 20 25 30 Lys Glu Gly Gln Asn Val Thr Leu Ser Cys Glu Gln Asn LeuAsn His 35 40 45 Asp Ala Met Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly LeuArg Leu 50 55 60 Ile Tyr Tyr Ser Gln Ile Val Asn Asp Phe Gln Lys Gly AspIle Ala 65 70 75 80 Glu Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Ser PhePro Leu Thr 85 90 95 Val Thr Ser Ala Gln Lys Asn Pro Thr Ala Phe Tyr LeuCys Ala Ser 100 105 110 Ser 113 amino acids amino acid linear peptideN-terminal unknown 8 Met Gly Pro Gln Leu Leu Gly Tyr Val Val Leu Cys LeuLeu Gly Ala 1 5 10 15 Gly Pro Leu Glu Ala Gln Val Thr Gln Asn Pro ArgTyr Leu Ile Thr 20 25 30 Val Thr Gly Lys Lys Leu Thr Val Thr Cys Ser GlnAsn Met Asn His 35 40 45 Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly LeuGly Leu Arg Gln 50 55 60 Ile Tyr Tyr Ser Met Asn Val Glu Val Thr Asp LysGly Asp Val Pro 65 70 75 80 Glu Gly Tyr Lys Val Ser Arg Lys Glu Lys ArgAsn Phe Pro Leu Ile 85 90 95 Leu Glu Ser Pro Ser Pro Asn Gln Thr Ser LeuTyr Phe Cys Ala Ser 100 105 110 Ser 113 amino acids amino acid linearpeptide N-terminal unknown 9 Met Gly Ile Arg Leu Leu Cys Arg Val Ala PheCys Phe Leu Ala Val 1 5 10 15 Gly Leu Val Asp Val Lys Val Thr Gln SerSer Arg Tyr Leu Val Lys 20 25 30 Arg Thr Gly Glu Lys Val Phe Leu Glu CysVal Gln Asp Met Asp His 35 40 45 Glu Asn Met Phe Trp Tyr Arg Gln Asp ProGly Leu Gly Leu Arg Leu 50 55 60 Ile Tyr Phe Ser Tyr Asp Val Lys Met LysGlu Lys Gly Asp Ile Pro 65 70 75 80 Glu Gly Tyr Ser Val Ser Arg Glu LysLys Glu Arg Phe Ser Leu Ile 85 90 95 Leu Glu Ser Ala Ser Thr Asn Gln ThrSer Met Tyr Leu Cys Ala Ser 100 105 110 Ser 113 amino acids amino acidlinear peptide N-terminal unknown 10 Met Gly Ile Arg Leu Leu Cys Arg ValAla Phe Cys Phe Leu Ala Val 1 5 10 15 Gly Leu Val Asp Val Lys Val ThrGln Ser Ser Arg Tyr Leu Val Lys 20 25 30 Arg Thr Gly Glu Lys Val Phe LeuGlu Cys Val Asp Met Asp His Glu 35 40 45 Asn Met Phe Trp Tyr Gln Arg GlnAsp Pro Gly Leu Gly Leu Arg Leu 50 55 60 Ile Tyr Phe Ser Tyr Asp Val LysMet Lys Glu Lys Gly Asp Ile Pro 65 70 75 80 Glu Gly Tyr Ser Val Ser ArgGlu Lys Lys Glu Arg Phe Ser Leu Ile 85 90 95 Leu Glu Ser Ala Ser Thr AsnGln Thr Ser Met Tyr Leu Cys Ala Ser 100 105 110 Ser 15 amino acids aminoacid linear peptide N-terminal unknown 11 Glu Gly Tyr Ser Val Ser ArgGlu Lys Lys Glu Ser Phe Pro Leu 1 5 10 15 15 amino acids amino acidlinear peptide N-terminal unknown 12 Glu Gly Tyr Ser Val Ser Arg Glu LysLys Glu Arg Phe Ser Leu 1 5 10 15 15 amino acids amino acid linearpeptide N-terminal unknown 13 Glu Gly Tyr Lys Val Ser Arg Lys Glu LysArg Asn Phe Pro Leu 1 5 10 15 15 amino acids amino acid linear peptideN-terminal unknown 14 Asp Gly Tyr Ser Val Ser Arg Ser Lys Thr Glu AspPhe Leu Leu 1 5 10 15 15 amino acids amino acid linear peptideN-terminal unknown 15 Asn Arg Phe Ser Pro Lys Ser Pro Asp Lys Ala HisLeu Asn Leu 1 5 10 15 15 amino acids amino acid linear peptideN-terminal unknown 16 Glu Gly Tyr Asp Ala Ser Arg Glu Lys Lys Ser SerPhe Ser Leu 1 5 10 15 15 amino acids amino acid linear peptideN-terminal unknown 17 Lys Gly Tyr Arg Val Ser Arg Lys Lys Arg Glu HisPhe Ser Leu 1 5 10 15 15 amino acids amino acid linear peptideN-terminal unknown 18 Asp Gly Tyr Lys Ala Ser Arg Pro Ser Gln Glu AsnPhe Ser Leu 1 5 10 15 15 amino acids amino acid linear peptideN-terminal unknown 19 Asp Gly Tyr Lys Ala Ser Arg Pro Ser Gln Lys GluPhe Ser Leu 1 5 10 15 18 amino acids amino acid linear peptideN-terminal unknown 20 Asp Gly Tyr Asn Val Ser Arg Leu Lys Lys Gln AsnPhe Leu Leu Gly 1 5 10 15 Leu Glu 8 amino acids amino acid linearpeptide N-terminal unknown 21 Ser Ser Asp Ser Ser Asn Thr Glu 1 5 8amino acids amino acid linear peptide N-terminal unknown 22 Ser Ser AspSer Gly Asn Thr Glu 1 5 9 amino acids amino acid linear peptideN-terminal unknown 23 Ser Gly Asp Ala Gly Gly Gly Tyr Glu 1 5 8 aminoacids amino acid linear peptide N-terminal unknown 24 Ser Ser Asp SerSer Asn Thr Glu 1 5 15 amino acids amino acid linear peptide N-terminalunknown 25 Cys Ala Ser Ser Asp Ser Ser Asn Thr Glu Val Phe Phe Gly Lys 15 10 15 15 amino acids amino acid linear peptide N-terminal unknown 26Cys Ala Ser Ser Asp Ser Gly Asn Thr Glu Val Phe Phe Gly Lys 1 5 10 15 17amino acids amino acid linear peptide N-terminal unknown 27 Cys Ala SerSer Asp Ser Gly Asn Val Leu Tyr Phe Gly Glu Gly Ser 1 5 10 15 Arg 9amino acids amino acid linear peptide N-terminal unknown 28 Ala Ser SerAsp Ser Ser Asn Thr Glu 1 5 20 amino acids amino acid linear peptideN-terminal unknown 29 Asp Met Gly His Gly Leu Arg Leu Ile His Tyr SerTyr Asp Val Asn 1 5 10 15 Ser Thr Glu Lys 20 21 amino acids amino acidlinear peptide N-terminal unknown 30 Asp Met Gly His Gly Leu Arg Leu IleHis Tyr Ser Tyr Asp Val Asn 1 5 10 15 Ser Thr Glu Lys Gly 20 11 aminoacids amino acid linear peptide N-terminal unknown 31 Arg Phe Gly AlaGly Thr Arg Leu Thr Val Lys 1 5 10 39 base pairs nucleic acid singlelinear DNA (genomic) unknown 32 CTCTGCAGCG GAGACCAGGG CGGCAATGAGCAGTTCTTC 39 11 amino acids amino acid linear peptide N-terminal unknown33 Ser Gly Asp Gln Gly Gly Asn Glu Gln Phe Phe 1 5 10 17 base pairsnucleic acid single linear DNA (genomic) unknown 34 GGAGACAGGA GGAAAGA17 21 base pairs nucleic acid single linear DNA (genomic) unknown 35GGCGACCAAG GCGGCAACGA A 21 21 base pairs nucleic acid single linear DNA(genomic) unknown 36 GGGGATCAGG GGGGGAATGA G 21 17 base pairs nucleicacid single linear DNA (genomic) unknown 37 GGTGACAGGT GGTAAGA 17 16amino acids amino acid linear peptide N-terminal unknown 38 Tyr Leu CysAla Ser Lys Asn Pro Thr Val Ser Tyr Gly Tyr Thr Phe 1 5 10 15 48 basepairs nucleic acid single linear DNA (genomic) unknown 39 TATCTCTGTGCCAGTAAAAA TCCCACGGTC TCCTATGGCT ACACCTTC 48 14 amino acids amino acidlinear peptide N-terminal unknown 40 Tyr Leu Cys Ala Ser Asp Asn Glu SerPhe Phe Gly Gln Gly 1 5 10 42 base pairs nucleic acid single linear DNA(genomic) unknown 41 TATCTCTGTG CCAGTGACAA CGAGAGTTTC TTTGGACAAG GC 4215 amino acids amino acid linear peptide N-terminal unknown 42 Tyr LeuCys Ala Ser Val Arg Asp Arg Arg Asn Tyr Gly Tyr Thr 1 5 10 15 45 basepairs nucleic acid single linear DNA (genomic) unknown 43 TATCTCTGTGCCAGTGTGAG GGACAGGAGA AACTATGGCT ACACC 45 15 amino acids amino acidlinear peptide N-terminal unknown 44 Tyr Leu Cys Ala Ser Ser Ser Ile AspSer Ser Tyr Glu Gln Tyr 1 5 10 15 45 base pairs nucleic acid singlelinear DNA (genomic) unknown 45 TATCTCTGTG CCAGTAGTAG TATAGACTCCTCCTACGAGC AGTAC 45 19 amino acids amino acid linear peptide N-terminalunknown 46 Val Pro Asn Gly Tyr Lys Val Ser Arg Pro Ser Gln Gly Asp PhePhe 1 5 10 15 Leu Thr Leu 16 base pairs nucleic acid single linear DNA(genomic) unknown 47 TGTACTGGTA CAAACA 16 16 base pairs nucleic acidsingle linear DNA (genomic) unknown 48 TTTTTTGGTA TCGTCA 16 24 basepairs nucleic acid single linear DNA (genomic) unknown 49 TCACAGATAGTAAATGACTT TCAG 24 18 base pairs nucleic acid single linear DNA(genomic) unknown 50 TCTCCACTCT GAAGATCC 18 18 base pairs nucleic acidsingle linear DNA (genomic) unknown 51 GATTTCCTCC TCACTCTG 18 18 basepairs nucleic acid single linear DNA (genomic) unknown 52 CAAGCTGTTCCCACCCGA 18 18 base pairs nucleic acid single linear DNA (genomic)unknown 53 CCAGAAGGTG GCCGAGAC 18 18 base pairs nucleic acid singlelinear DNA (genomic) unknown 54 GCGGCTGCTC AGGCAGTA 18 18 base pairsnucleic acid single linear DNA (genomic) unknown 55 CGACCTCGGG TGGGAACA18 15 base pairs nucleic acid single linear DNA (genomic) unknown 56GAAGTCTGGA ACAGC 15 15 base pairs nucleic acid single linear DNA(genomic) unknown 57 GACGTCTGGA ACAGC 15 15 base pairs nucleic acidsingle linear DNA (genomic) unknown 58 GTAGTATGTT CTGCA 15 15 base pairsnucleic acid single linear DNA (genomic) unknown 59 GTAGTGTGTT CTGCA 1524 base pairs nucleic acid single linear DNA (genomic) unknown 60CTCCTCGAGC AGAGGCGGGC CGCG 24 24 base pairs nucleic acid single linearDNA (genomic) unknown 61 TTCCTCGAGG ACAGGCGCGC CGCG 24 24 base pairsnucleic acid single linear DNA (genomic) unknown 62 CTCCTCGAGCAGAAGCGGGG CCGG 24 24 base pairs nucleic acid single linear DNA(genomic) unknown 63 CTCCTCGAGC AGAAGCGGGC CGCG 24 24 base pairs nucleicacid single linear DNA (genomic) unknown 64 CTCCTCGAGC AGAGGCGGGC CGAG24 24 base pairs nucleic acid single linear DNA (genomic) unknown 65ATCCTCGAAG ACGAGCGGGC CGCG 24 24 base pairs nucleic acid single linearDNA (genomic) unknown 66 ATCCTCGAGG ACAGGCGGGG CCAG 24 24 base pairsnucleic acid single linear DNA (genomic) unknown 67 TTCCTCGAAGACAGGCGGGC CCTG 24 24 base pairs nucleic acid single linear DNA(genomic) unknown 68 TTCCTCGAGC GGAGGCGGGC CGAG 24 24 base pairs nucleicacid single linear DNA (genomic) unknown 69 ATCCTCGAGC AGGCGCGGGC CGCG24 24 base pairs nucleic acid single linear DNA (genomic) unknown 70CTCCTCGAGC AGAAGCGGGG CCAG 24 24 base pairs nucleic acid single linearDNA (genomic) unknown 71 CTCCTCGAGC GGAGGCGGGC CGAG 24 21 amino acidsamino acid linear peptide N-terminal unknown 72 Asp Pro Gly Leu Gly LeuArg Leu Ile Tyr Phe Ser Tyr Asp Val Lys 1 5 10 15 Met Lys Glu Lys Gly 2021 amino acids amino acid linear peptide N-terminal unknown 73 Asp ProGly Leu Gly Leu Arg Gln Ile Tyr Tyr Ser Met Asn Val Glu 1 5 10 15 ValThr Asp Lys Gly 20 21 amino acids amino acid linear peptide N-terminalunknown 74 Asp Pro Gly Gln Gly Leu Arg Leu Ile Tyr Tyr Ser Gln Ile ValAsn 1 5 10 15 Lys Phe Gln Lys Gly 20 101 amino acids amino acid linearpeptide N-terminal unknown 75 Phe Leu Ala Val Gly Leu Val Asp Val LysVal Thr Gln Ser Ser Arg 1 5 10 15 Tyr Leu Val Lys Arg Thr Gly Glu LysVal Phe Leu Glu Cys Val Gln 20 25 30 Asp Met Asp His Glu Asn Met Phe TrpTyr Arg Gln Asp Pro Gly Leu 35 40 45 Gly Leu Arg Leu Ile Tyr Phe Ser TyrAsp Val Lys Met Lys Glu Lys 50 55 60 Gly Asp Ile Pro Glu Gly Tyr Ser ValSer Arg Glu Lys Lys Glu Arg 65 70 75 80 Phe Ser Leu Ile Leu Glu Ser AlaSer Thr Asn Gln Thr Ser Met Tyr 85 90 95 Leu Cys Ala Ser Ser 100

We claim:
 1. A method for eliciting an immune response in an individualsuffering from rheumatoid arthritis (RA), comprising administeringdirectly into muscle tissue of said individual a plasmid vectorcomprising a promoter operably linked to a nucleic acid sequenceencoding a single chain T cell receptor variable beta 3, 14 or 17peptide, or fragments thereof, wherein said nucleic acid sequence isexpressed in said muscle tissue at a level sufficient to elicit animmune response against the encoded peptide in said individual.
 2. Themethod of claim 1, wherein said nucleic acid sequence encodes a CDR2region of said β-chain variable region.
 3. The method of claim 1,wherein said nucleic acid sequence encodes a superantigen binding regionof said β-chain variable region, or degenerative sequences thereof. 4.The method of claim 1, wherein said nucleic acid vector is administeredwith a pharmaceutically acceptable medium.
 5. A composition, comprisinga plasmid vector in a pharmaceutically acceptable medium for elicitingan immune response in an individual suffering from rheumatoid arthritis,said plasmid vector comprises a promoter operably linked to a nucleicacid encoding a single chain T cell receptor variable beta 3, 14 or 17peptide.
 6. The composition of claim 5, wherein said nucleic acidsequence encodes a CDR2 region of said β-chain variable region.
 7. Thecomposition of claim 5, wherein said nucleic acid sequence encodes asuperantigen binding region of said β-chain variable region, ordegenerative sequences thereof.